Nanostructured gels capable of controlled release of entrapped agents

ABSTRACT

Self-assembled gel compositions including a gelator, e.g., an enzyme-cleavable gelator, e.g., having a molecular weight of 2500 or less, are described. The self-assembled gel compositions can encapsulate one or more agents. Methods of making the self-assembled gel compositions, and methods of drug delivery using the self-assembled gel compositions are also described.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/386,268, filed on Sep. 24, 2010, and U.S. Provisional Application No.61/466,753, filed on Mar. 23, 2011. The contents of each of these priorapplications are incorporated herein by reference in their entireties.

TECHNICAL FIELD

This invention relates to self-assembled gels including gelators havinga relatively small molecular weight, and more particularly toself-assembled gels including generally recognized as safe (GRAS)gelators.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted on Aug. 23, 2016, as a text file named“BWH_21124_MIT_15218_CON_ST25.txt,” created on Jun. 28, 2016, and havinga size of 1,066 bytes is hereby incorporated by reference pursuant to 37C.F.R § 1.52(e)(5).

BACKGROUND

Local delivery of drugs can provide high local drug concentration whileminimizing systemic toxicity, which can often be observed with oraldosing. However, as local depots are generally administered lessfrequently and include an initial burst followed by a continuousrelease, to maximize efficiency of therapy, it is desirable that a drugis only released when needed.

Delivering drugs to patients in a safe, effective, and compliant manneris a major challenge for the treatment of many types of disease. Theability of drugs to reach target tissues from the point of oraladministration can be limited by multiple barriers including enzymaticand acidic degradation in the stomach, absorption across the intestinalepithelium, hepatic clearance, and nonspecific uptake. Effective oraldosing to achieve high concentrations of drugs within specific tissueswhile minimizing systemic toxicity can present a significant challenge.Conventional polymeric drug delivery systems such as implants,injectable microspheres, and patches are used by tens of millions ofpeople annually, yet often produce a sharp initial increase inconcentration to a peak above the therapeutic range, followed by a fastdecrease in concentration to a level below the therapeutic range.Additionally, noncompliance with oral medication is a leading cause ofhospitalizations.

The holy grail of drug delivery is an autonomous system that can titratethe amount of drug released in response to a biological stimulus,thereby ensuring that the drug is released when needed at atherapeutically relevant concentration. Such a system can rapidlyrelease drug in response to fluctuations due to the severity of disease(this is often reflected by the local inflammatory state),patient-to-patient variability, and environmental factors.

SUMMARY

The disclosure relates, at least in part, to self-assembled gelcompositions including one or more generally recognized as safe (GRAS)gelators. Substances and agents such as small molecular agents, drugs,drug-candidates, vitamins, proteins, dyes and sensors can beencapsulated within the assembled structures. The encapsulated substanceor substances can be subsequently delivered through hydrolytic or otherforms of degradation of the self-assembled gels or in response to anexternal stimulus, such as a specific enzyme. In some embodiments, theself-assembled gels can be formed of one or more amphiphilic gelators,which can encapsulate one or more different agents (e.g., therapeuticagents). The new amphiphilic gelators can act in synergy with theencapsulated agent, such that a therapeutic effect of the encapsulatedagent is enhanced compared to a non-encapsulated agent. In someembodiments, self-assembled gels can encapsulate and release two or moredifferent agents that can act synergistically to achieve enhancedefficacy. In some embodiments, self-assembled gels can include vitaminsor vitamin derivatives in combination with either another vitaminderivative or a GRAS gelator. The self-assembled gels can increasestability of agents, such as encapsulated therapeutic agents and/orvitamins, e.g., from photo/ultra-violet degradation, and can deliverhigh concentrations of vitamins or GRAS agents.

The disclosure also relates, at least in part, to self-assembledhydrogel compositions including an enzyme-cleavable GRAS gelator, suchas a GRAS gelator including a molecular weight of 2,500 or less. Thehydrogel compositions can self-assemble under specific assemblyconditions. Hydrogels can offer advantages such as the ability tohydrate in aqueous conditions and enhanced biological compatibility, andcan be well suited for biological administration (e.g., implantation ofwet hydrogels). Furthermore, the disclosure relates, at least in part,to organogels formed of GRAS gelators such as ascorbyl alkanoate,sorbitan alkanoate, triglycerol monoalkanoate, sucrose alkanoate, and/orglycocholic acid.

In one aspect, the disclosure features self-assembled gel compositionsincluding enzyme-cleavable, generally recognized as safe (GRAS) firstgelators having a molecular weight of 2500 or less. The GRAS firstgelators can include ascorbyl alkanoate, sorbitan alkanoate, triglycerolmonoalkanoate, sucrose alkanoate, glycocholic acid, and/or anycombination thereof. The GRAS first gelators can self-assemble into gelsincluding nanostructures.

In another aspect, the disclosure features self-assembled gelcompositions capable of controlled release of agents. The self-assembledgel compositions include enzyme-cleavable, generally recognized as safe(GRAS) first gelators having a molecular weight of 2500 or less; and oneor more agents, e.g., any agents as described herein. The GRAS firstgelators can include ascorbyl alkanoate, sorbitan alkanoate, triglycerolmonoalkanoate, sucrose alkanoate, glycocholic acid, and/or anycombination thereof, and can self-assemble into gels includingnanostructures. The agents can be encapsulated within or between thenanostructures, can be non-covalently bonded to the nanostructures, orboth.

In yet another aspect, the disclosure features methods of formingself-assembled gel compositions. The methods include combiningenzyme-cleavable generally recognized as safe (GRAS) gelators having amolecular weight of 2500 or less and solvents to form a mixture; heatingor sonicating the mixture; stirring or shaking the mixture for a timesufficient to form a homogeneous solution; and cooling the homogenoussolution for a time sufficient to enable the formation of self-assembledgel compositions. The GRAS gelators can include ascorbyl alkanoate,sorbitan alkanoate, triglycerol monoalkanoate, sucrose alkanoate,glycocholic acid, and/or any combination thereof. In some embodiments,the methods further include lyophilizing self-assembled gels to formxerogels.

In another aspect, the disclosure features methods of forming aself-assembled gel composition. The methods include combiningenzyme-cleavable generally recognized as safe (GRAS) first gelatorshaving a molecular weight of 2500 or less and second gelators to form amixture; heating or sonicating the mixture; stirring or shaking themixture for a time sufficient to form a homogeneous solution; andcooling the homogenous solution for a time sufficient to enable theformation of self-assembled gel compositions. The GRAS first gelatorscan include ascorbyl alkanoate, sorbitan alkanoate, triglycerolmonoalkanoate, sucrose alkanoate, glycocholic acid, and/or anycombination thereof, and second gelators can include alpha tocopherolacetate, retinyl acetate, and/or retinyl palmitate. In some embodiments,the methods further include lyophilizing the self-assembled gel to forma xerogel.

In a further aspect, the disclosure features self-assembled gelcompositions including amphiphilic 3-aminobenzamide derivatives having amolecular weight of 2500 or less. The amphiphilic 3-aminobenzamindederivatives can self-assemble into gels comprising nanostructures. Theself-assembled gel compositions can further include an agent, and theagent can be encapsulated within or between the nanostructures ornon-covalently bonded to the nanostructures.

In yet a further aspect, the disclosure features self-assembled gelcompositions including an enzyme-cleavable, generally recognized as safe(GRAS) first gelator having a molecular weight of 2500 or less and anon-independent second gelator. The first gelator and thenon-independent second gelator each independently can have aconcentration of from 0.01 to 20 percent by weight per gel volume. TheGRAS first gelator can include ascorbyl alkanoate, sorbitan alkanoate,triglycerol monoalkanoate, sucrose alkanoate, glycocholic acid, and anycombination thereof. The non-independent second gelator can includealpha tocopherol acetate, retinyl acetate, and retinyl palmitate.

Embodiments of the above-mentioned aspects can have one or more of thefollowing features.

In some embodiments, the ascorbyl alkanoates include ascorbyl palmitate,ascorbyl decanoate ascorbyl laurate, ascorbyl caprylate, ascorbylmyristate, ascorbyl oleate, and/or any combination thereof. For example,the ascorbyl alkanoates can include ascorbyl palmitate. In someembodiments, the sorbitan alkanoates include sorbitan monostearate,sorbitan decanoate, sorbitan laurate, sorbitan caprylate, sorbitanmyristate, sorbitan oleate, and/or any combination thereof. For example,the sorbitan alkanoate can include sorbitan monostearate. In someembodiments, the triglycerol monoalkanoates include triglycerolmonopalmitate, triglycerol monodecanoate, triglycerol monolaurate,triglycerol monocaprylate, triglycerol monomyristate, triglycerolmonostearate, triglycerol monooleate, and/or any combination thereof.For example, the triglycerol monoalkanoates include triglycerolmonopalmitate. In some embodiments, the sucrose alkanoates includesucrose palmitate, sucrose decanoate, sucrose laurate, sucrosecaprylate, sucrose myristate, sucrose oleate, and/or any combinationthereof. For example, the sucrose alkanoates can include sucrosepalmitate. In some embodiments, the GRAS first gelators includeglycocholic acid.

In some embodiments, the self-assembled gel compositions includenon-independent second gelators that can include alpha tocopherolacetate, retinyl acetate, and/or retinyl palmitate. The non-independentsecond gelators can co-assemble with the GRAS first gelators to form theself-assembled gels.

The self-assembled gel compositions can be solvent-free. When theself-assembled gels are solvent free, the gels can include from 0.5(e.g., from one, from two, from three, from five, from 10, from 15, orfrom 20) to 25 (e.g., to 20, to 15, to 10, to five, to three, to two, orto one) percent by weight of the GRAS or non-GRAS first gelator and from75 (e.g., from 80, from 85, from 90, from 95, from 97, from 98, or from99) to 99.5 (e.g., to 99, to 98, to 97, to 95, to 90, to 85, or to 80)percent by weight of the non-independent second gelator. In someembodiments, the gel compositions can include independently from 0.01(e.g., from 0.05, from 0.5, from one, from two, from three, from five,from 10, or from 15) to 25 percent (to 20, to 15, to 10, to five, tothree, to two, to one, to 0.5, to 0.05) by weight per gel volume of theGRAS and/or of the non-independent second gelator.

In some embodiments, the self-assembled gel compositions can include apolar or non-polar solvent, such as water, benzene, toluene, carbontetrachloride, acetonitrile, glycerol, 1,4-dioxane, dimethyl sulfoxide,ethylene glycol, methanol, chloroform, hexane, acetone, N, N′-dimethylformamide, ethanol, isopropyl alcohol, butyl alcohol, pentyl alcohol,tetrahydrofuran, xylene, mesitylene, and/or any combination thereof.When the self-assembled gels include a solvent, the gels can includebetween 0.01 and 18 (e.g., between 0.05 and 18, between 0.01 and 15,between 0.05 and 15, between 0.1 and 15, between 0.5 and 15, between oneand 15, or between one and 10) weight/volume percent of one or moregenerally recognized as safe gelators in the solvent.

In some embodiments, the nanostructures can include lamellae formed ofthe enzyme-cleavable GRAS first gelators. The agents (e.g., hydrophobicor hydrophilic) can be encapsulated between the lamellae. As an example,the agents can include a steroid, an anti-inflammatory agent, achemotherapeutic, a PARP-inhibitor, a polypeptide, a nucleic acid, apolynucleotide, a polyribonucleotide, an anti-pain agent, ananti-pyretic agent, an anti-depression agent, a vasodilator, avasoconstrictor, an immune-suppressant, a tissue regeneration promoter,a vitamin, a small interfering RNA, a polymer selected from the groupconsisting of poly(ethylene glycol), poly(ethylene oxide), hyaluronicacid, chitosan, carboxy methylcellulose, poly(ethylene glycol)di-acrylate, and poly(glycerol-co-sebasate acrylate), any derivativethereof, and/or any combination thereof. In some embodiments, when theself-assembled gel compositions include two or more agents, at least oneagent potentiates an efficacy of one or more remaining agents.

In some embodiments, the amphiphilic 3-aminobenzamide derivatives can astructure of formula (I):

-   -   wherein

A is CR₁R₂ or O, wherein R₁ and R₂ are each independently H or halogen;

E is C₁₋₂ alkyl, C₁₋₂ haloalkyl, or absent;

D is selected from the group consisting of C₃₋₂₀ alkyl, C₂₋₆ alkenyl,aryl, C₃₋₂₀ cycloalkyl, wherein each are optionally substituted with 1,2, 3, or 4 groups selected from the group consisting of C₁₋₄ alkoxy,C₁₋₈ alkyl, halo, C₁₋₈ haloalkyl, and nitro.

In some embodiments, the amphiphilic 3-aminobenzamide derivatives can beselected from:

In some embodiments, when applied to a biological system, theamphiphilic 3-aminobenzamide derivatives can potentiates an efficacy(e.g., enhance the efficacy, and/or act synergistically with) of agents,such as chemotherapeutic agents (e.g., temozolomide, carmustine(bis-chloroethylnitrosourea), camptothecin, or and/paclitaxel).

When applied to a biological system, the self-assembled gel compositionsdescribed herein can provide controlled release of agents. The gelcompositions can be adapted to be controllably disassembled.

In some embodiments, the self-assembled gel compositions are lubriciousand/or have recoverable rheological properties. In some embodiments, theself-assembled gel compositions have an elastic modulus of from 10 to10,000 Pascal and a viscous modulus of from 10 to 10,000 Pascal.

Embodiments and/or aspects can provide one or more of the followingadvantages.

The self-assembled gel compositions can enhance the stability andfacilitate delivery of encapsulated agents or of gelators forming thegel. The self-assembled gel compositions can provide controlled releaseof an encapsulated agent, for example, upon exposure to a specificstimulus. The self-assembled gel compositions can act in synergy with anencapsulated agent, such that the efficacy of the agent is enhanced. Ingeneral, the gel compositions are relatively stable and easy tosynthesize.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of a self-assembling gelcomposition, an agent encapsulation process, and a gel disassemblyprocess.

FIGS. 2A and 2B are graphs showing agent release from an ascorbicpalmitate (“Asc-Pal”) self-assembled gel fibers in response to lipase,MMP-2, and MMP-9 at 37° C. in vitro. (A) Enzyme was added on day 0, andrelease kinetics were continuously monitored. (B) After 4 days of enzymeaddition, media were changed to remove the enzyme (dotted arrow); on day11, fresh enzyme was added (solid arrow), triggering the release of dye.

FIGS. 3A and 3B are graphs showing (A) synovial fluid collected fromarthritis patients mediated fiber disassembly and dye release over a15-day period, whereas dye was not released from gels incubated withPBS. (B) Gel fibers were incubated with synovial lysates prepared fromankle tissue of arthritic mice with and without protease inhibitors.

FIGS. 4A and 4B are graphs showing dynamic rheology of Asc-Pal fibroushydrogels assessed with a parallel-plate rheometer. (A) Storage modulus,GO, and viscous modulus, G″, over a frequency range of 0-12 rad/s; (B)Frequency sweeps conducted before/after multiple cycles of a high shearstress to measure GO.

FIG. 5 is a graphical representation of a self-assembling gel includinga 3-aminobenzamide derivative and optionally an encapsulatedtomozolomide.

FIG. 6 is a graph showing increased stability of temozolomide (TMZ) ingel and xerogel formed from an Abz-Chol amphiphile.

FIG. 7A is a graph showing a release of tomozolomide from gel fibers andxerogel fibers into DPBS at 37° C. FIG. 7B is a graph showing areleaseover first 12 hours.

FIG. 8 is a graph showing a cumulative release of tomozolomide in DPBSat 37° C. from a transwell formulation, measured by high performanceliquid chromatograph (“HPLC”).

FIG. 9 is a graph showing controlled release of camptothecin fromself-assembled hydrogel fibers of SMS (6%, wt/v) in the absence andpresence of esterase enzyme (10,000 units) at 37° C.

FIGS. 10A and 10B are graphs showing viability of (A) glioma cells (G55)and (B) fibroblasts (NIH3T3) in the presence ofcamptothecin-encapsulated self-assembled SMS gel fibers.

FIGS. 11A and 11B are graphs summarizing cytotoxicity of glioblastomacancer cell lines ((A) G55 and (B) U87) using chemotherapeutic agent(CPT) and PARP-inhibitor (AGO14699) loaded Sorbitan Monostearate gels,individual and combination.

FIG. 12 is a graph showing controlled release of TA from self-assembledSMS hydrogels (6%, wt/v) in the absence and presence of esterase enzyme(10,000 units) at 37° C.

FIGS. 13A-13 B are graphs showing A) encapsulation efficiency and B)loading efficiency of Asc-Pal hydrogels (20% ethanol v/v) at differentconcentration of Asc-Pal amphiphile (1-5% wt/v). In all samples, 2 mg ofdexamethasone was used as starting concentration for encapsulation.

FIG. 14A-14B are graphs showing controlled release of dexamethasone fromself-assembled hydrogels (ethanol:water, 1:3) of Asc-Pal (5%, wt/v) inthe absence and presence of esterase enzyme (10,000 units) at 37° C.Controlled release was examined for A) 2 mg and B) 4 mg of dexamethasoneencapsulated within Asc-Pal hydrogels. Similar release profile has beenobserved from both gels.

FIG. 15A-15B are graphs showing enzyme responsive degradation of Asc-Pal(3 and 6% wt/v) hydrogels to release ascorbic acid from A) low and B)high loading of dexamethasone. Release of ascorbic acid was measuredwith and without lipase enzyme (10,000 units) at 37° C. In the presenceof enzyme, Asc-Pal was cleaved rapidly releasing ascorbic acid (within 2hours), whereas absence of enzyme did not resulted in the presence ofsignificant concentrations of ascorbic acid. Ascorbic acid is unstableand degrades rapidly in PBS at 37° C., thus its concentration decreasesover time.

FIG. 16 is a graph showing release of dexamethasone from Asc-Palhydrogels that were doped with an excipient glycocholic acid. 1 mg ofglycocholic acid was added during the preparation of hydrogels. 3 and 6%(wt/v) of amphiphile was used to prepare gels. Release of dexamethasonewas been performed in the absence (3% APDGC-NE and 6% APDCG-NE) and inthe presence of (3% APDGC-WE and 6% APDCG-WE) lipase enzyme (10,000units) at 37° C.

FIG. 17 is a graph showing on-demand release of dexamethasone fromdexamethasone-palmitate encapsulated self-assembled hydrogels (20% (v/v)DMSO in water) of Asc-Pal (8%, wt/v) in the absence and presence ofesterase enzyme (10,000 units) at 37° C. From day-0 to day-11 gels wereincubated in PBS, on day-11 lipase was added (arrow) that triggeredrelease of dexamethasone.

FIG. 18 is a graph showing controlled release of indomethacin fromself-assembled hydrogel of Asc-Pal (6%, wt/v) in the absence andpresence of esterase enzyme (10,000 units) at 37° C.

FIG. 19 is a graph showing quantification of DNA in SMS hydrogel fibers.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

There exist broad implications for achieving an on demand drug deliveryapproach for the treatment of tissue defects and multiple diseases. Oneapproach toward this goal is the design of compounds tailored to releasedrugs in response to the local expression of enzymes that correlate withthe level of inflammation. Inflammatory conditions that arecharacterized by the generation of enzymes that destroy extracellularconnective tissue—such as occurs in rheumatoid arthritis (RA) and woundhealing comprise a particularly attractive first application. Bytargeting other disease-associated enzyme pathways, this platform canhave broad applicability for diseases such as cancer, ocular disease,oral disease, gastrointestinal disease, and cardiovascular disease.

Hydro- or organo-gel compositions as described herein consist ofself-assembled macromolecular, nanostructure networks with a liquidfilling the interstitial space of the network. The network holds theliquid in place through its interaction forces and so gives the gelsolidity and coherence, but the gel is also wet and soft and capable ofundergoing some extent of deformation. The gel state is neither solidnor liquid, but has some features of both. Self-assembly has been usedto develop molecularly defined and functional materials, includinghydrogels.

Self-assembled hydrogel compositions can be formulated in a variety ofphysical forms, including microparticles, nanoparticles, coatings andfilms. As a result, hydrogels are commonly used in clinical practice andexperimental medicine for a wide range of applications, including tissueengineering and regenerative medicine, diagnostics, cellularimmobilization, separation of biomolecules or cells and barriermaterials to regulate biological adhesions. Hydrogel compositions areappealing for biological applications because of their high watercontent and biocompatibility.

Definitions

The term “C₂₋₆ alkenyl” denotes a group containing 2 to 6 carbonswherein at least one carbon-carbon double bond is present, someembodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, andsome embodiments have 2 carbons. Both E and Z isomers are embraced bythe term “alkenyl.” Furthermore, the term “alkenyl” includes di- andtri-alkenyls. Accordingly, if more than one double bond is present thenthe bonds may be all E or Z or a mixtures of E and Z. Examples of analkenyl include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl,3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl,2,4-hexadienyl and the like.

The term “C₁₋₄ alkoxy” as used herein denotes a group alkyl, as definedherein, attached directly to an oxygen atom. Examples include methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy, iso-butoxy,sec-butoxy and the like.

The term “alkyl” denotes a straight or branched carbon group containing3 to 20 carbons, and some embodiments are 1 to 8 carbons. Examples of analkyl include, but not limited to, methyl, ethyl, n-propyl, iso-propyl,n-butyl, sec-butyl, iso-butyl, t-butyl, pentyl, iso-pentyl, t-pentyl,neo-pentyl, 1-methylbutyl (i.e., —CH(CH₃)CH₂CH₂CH₃), 2-methylbutyl(i.e., —CH₂CH(CH₃)CH₂CH₃), n-hexyl, lauryl, decanoyl, palmityl,caprylyl, myristyl, oleyl, stearyl and the like.

The term “C₁₋₂ alkylene” refers to a C₁₋₂ divalent straight carbongroup. In some embodiments C₁₋₂ alkylene refers to, for example, —CH₂—,—CH₂CH₂—, and the like.

The term “aryl” denotes an aromatic ring group containing 6 to 14 ringcarbons. Examples include phenyl, naphthyl, and fluorenyl.

The term “C₃₋₂₀ cycloalkyl” denotes a saturated ring group containing 3to 20 carbons; some embodiments contain 3 to 17 carbons; someembodiments contain 3 to 4 carbons. Examples include cyclopropyl,cyclobutyl, cyclopentyl, cyclopenyl, cyclohexyl, cycloheptyl and thelike.

The term “halogen” or “halo” denotes to a fluoro, chloro, bromo or iodogroup.

The term “C₁₋₂ haloalkyl” denotes an C₁₋₂ alkyl group, defined herein,wherein the alkyl is substituted with one halogen up to fullysubstituted and a fully substituted C₁₋₂ haloalkyl can be represented bythe formula C_(n)L_(2n+1) wherein L is a halogen and “n” is 1, or 2;when more than one halogen is present then they may be the same ordifferent and selected from the group consisting of F, Cl, Br and I,preferably F. Examples of C₁₋₂ haloalkyl groups include, but not limitedto, fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl,2,2,2-trifluoroethyl, pentafluoroethyl and the like.

The term “C₁₋₈ haloalkyl” denotes an C₁₋₂ alkyl group, defined herein,wherein the alkyl is substituted with one halogen up to fullysubstituted and a fully substituted C₁₋₂ haloalkyl can be represented bythe formula C_(n)L_(2n+1) wherein L is a halogen and “n” is 1, 2, 3, 4,5, 6, 7, or 8; when more than one halogen is present then they may bethe same or different and selected from the group consisting of F, Cl,Br and I, preferably F.

The term “nitro” refers to the group —NO₂.

A “non-independent gelator” is a molecule that cannot, by itself, form aself-assembled gel, but can form an integral part of a self-assembledgel in the presence of another gelator that can promote gelation of thenon-independent gelator, such as a GRAS gelator. The non-independentgelator forms part of the gel structure (e.g., a lamellar structure)with the gelator that promotes gelation of the non-dependent gelator.The non-independent gelator can be amphiphilic, having a hydrophilicgroup attached to a hydrophobic group, which can co-assemble with otherhydrophilic and hydrophobic groups of an accompanying gelator moleculeto form the gel structure.

“Hydrogels,” as known to those of skill in the art, are 3-D networks ofmolecules typically covalently (e.g., polymeric hydrogels) ornon-covalently (e.g., self-assembled hydrogels) held together wherewater is the major component (usually greater than 80%). Gels can beformed via self-assembly of gelators or via chemical crosslinking ofgelators. Water-based gelators can be used to form hydrogels, whereasorganogelators are gelators that form gels (organogels) in solventswhere organic solvents are the major component.

“Organogels,” as known to those of skill in the art, are 3-D networks ofmolecules typically covalently (e.g., polymeric hydrogels) ornon-covalently (e.g., self-assembled hydrogels) held together where anorganic solvent is the major component (usually greater than 80%). Gelscan be formed via self-assembly of gelators or via chemical crosslinkingof gelators.

“Gelators,” as known to those of skill in the art, are molecules thatcan self-assemble through non-covalent interactions, such ashydrogen-bonding, van der Waals interactions, hydrophobic interactions,ionic interactions, pi-pi stacking, or combinations thereof, in one ormore solvents. The gelators can form a gel by rigidifying the solventthrough, for example, capillary forces. Gelators can includehydrogelators (e.g., gelators that form hydrogels) and organogelators(e.g, gelators that form organogels). In some embodiments, gelators canform both hydrogels and organogels.

Self-Assembled Gel Compositions

Generally, self-assembled gel compositions can include an amphiphilicgelator having a molecular weight of 2500 or less, such as anenzyme-cleavable, generally recognized as safe (GRAS) gelator having amolecular weight of 2500 or less. The generally recognized as safegelator can include any agent listed on the FDA's GRAS list. Forexample, the GRAS gelator can include, but is not limited to, agentsthat are generally recognized, among experts qualified by scientifictraining and experience to evaluate their safety, as having beenadequately shown through scientific procedures (or, in the case of asubstance used in food prior to Jan. 1, 1958, through either scientificprocedures or through experience based on common use in food) to besafe.

Without wishing to be bound by theory, it is believed that whenamphiphilic molecules self-assemble in a solvent, hydrophobic andhydrophilic portions of the gelator molecules can interact to formlamellae of gelator molecules. In some embodiments, when the gels arehydrogels, the hydrophobic portions of gelators are located in the innerregions of a given lamella, and hydrophilic portions are located at theouter surfaces of the lamella. In some embodiments, when the gels areorganogels, the hydrophobic portions of gelators are located in theouter regions of a given lamella, and hydrophilic portions are locatedat the inner surfaces of the lamella. The lamella can have a width offrom about three (e.g., from about four) to about five (e.g., to aboutfour) nanometers and a length of several microns (e.g., one micron, twomicrons, three microns, four microns, five microns, ten microns, twentymicrons, or twenty five microns) or more. Several tens or hundreds ofsuch lamellae can bundle together to form nanostructures, such as fibersand sheet-like structures. In some embodiments, the nanostructures caninclude nanoparticles, micelles, liposome vesicles, fibers, and/orsheets. In some embodiments, The nanostructures can have a minimumdimension (e.g., a thickness, a width, or a diameter) of 2 nm or more(e.g., 50 nm or more, 100 nm or more, 150 nm or more, 200 nm or more,250 nm or more, 300 nm or more, 350 nm or more) and/or 400 nm or less(e.g., 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less,150 nm or less, 100 nm or less, or 500 nm or less). In some embodiments,the nanostructures (e.g, fibers, sheets) can have a length and/or widthof several microns (e.g., one micron, two microns, three microns, fourmicrons, five microns, ten microns, twenty microns, or twenty fivemicrons) or more. The nanostructures can aggregate into networks, and/orbe in the form of a liquid crystal, emulsion, fibrillar structure, ortape-like morphologies. When the nanostructures are in the form offibers, the fibers can have a diameter of about 2 nm or more, and canhave lengths of hundreds of nanometers or more. In some embodiments, thefibers can have lengths of several microns (e.g., one micron, twomicrons, three microns, four microns, five microns, ten microns, twentymicrons, or twenty five microns) or more.

In some embodiments, the GRAS gelators can include ascorbyl alkanoate,sorbitan alkanoate, triglycerol monoalkanoate, sucrose alkanoate,glycocholic acid, or any combination thereof. The alkanoate can includea hydrophobic C₁-C₂₂ alkyl (e.g., acetyl, ethyl, propyl, butyl, pentyl,caprylyl, capryl, lauryl, myristyl, palmityl, stearyl, arachidyl, orbehenyl) bonded via a labile linkage (e.g., an ester linkage) to anascorbyl, sorbitan, triglycerol, or sucrose molecule. For example, theascorbyl alkanoate can include ascorbyl palmitate, ascorbyl decanoate,ascorbyl laurate, ascorbyl caprylate, ascorbyl myristate, ascorbyloleate, or any combination thereof. The sorbitan alkanoate can includesorbitan monostearate, sorbitan decanoate, sorbitan laurate, sorbitancaprylate, sorbitan myristate, sorbitan oleate, or any combinationthereof. The triglycerol monoalkanoate can include triglycerolmonopalmitate, triglycerol monodecanoate, triglycerol monolaurate,triglycerol monocaprylate, triglycerol monomyristate, triglycerolmonostearate, triglycerol monooleate, or any combination thereof. Thesucrose alkanoate can include sucrose palmitate, sucrose decanoate,sucrose laurate, sucrose caprylate, sucrose myristate, sucrose oleate,or any combination thereof. In some embodiments, the GRAS gelatorsinclude ascorbyl palmitate, sorbitan monostearate, triglycerolmonopalmitate, sucrose palmitate, or glycocholic acid.

In some embodiments, the self-assembled gel compositions can include oneor more non-independent second gelators, such as a vitamin derivative,that is or are different from a GRAS gelator. For example,self-assembling gels can be formed of vitamins or vitamin derivatives incombination with another vitamin derivative, a GRAS gelator, or anon-GRAS gelator. The non-independent gelators cannot assemble into agel by itself. However, the use of a first gelator, such as a GRAS firstgelator, can promote the gelation of a non-independent second gelator,such that both the first and second gelators can co-assemble into a geland can both be integrated into the gel structure (e.g., lamellar,micellar, vesicular, or fibrous structures), where neither gelcomponents are merely encapsulated by the gel. In the case ofnon-independent vitamin gelator derivatives, the resulting gels canincrease resistance to photo/ultra-violet degradation of vitamins anddeliver high concentrations of vitamins or GRAS gelators.

In some embodiments, the non-independent second gelators include aliquid amphiphile. For example, the non-independent second gelators caninclude alpha tocopherol acetate, retinyl acetate, and/or retinylpalmitate. When the non-independent second gelators are liquidamphiphiles, the resulting gels can include a solvent, or besolvent-free. When the gels are solvent-free, the gels can include from0.5 (e.g., from one, from two, from three, from five, from 10, from 15,or from 20) to 25 (e.g., to 20, to 15, to 10, to five, to three, to two,or to one) percent by weight of the GRAS or non-GRAS first gelator andfrom 75 (e.g., from 80, from 85, from 90, from 95, from 97, from 98, orfrom 99) to 99.5 (e.g., to 99, to 98, to 97, to 95, to 90, to 85, or to80) percent by weight of the non-independent second gelator. When thegels includes a solvent, the gels can include, independently, from 0.01(e.g., from 0.05, from 0.5, from one, from two, from three, from five,from 10, or from 15) to 25 percent (to 20, to 15, to 10, to five, tothree, to two, to one, to 0.5, to 0.05) by weight per gel volume of theGRAS or non-GRAS first gelator and the non-independent second gelator.The resulting gels can be relatively stable and provide enhancedstability of the gel constituents (e.g., vitamin E and/or vitamin Aderivatives).

In some embodiments, the self-assembled gel compositions include asolvent. Examples of solvents include water, benzene, toluene, carbontetrachloride, acetonitrile, glycerol, 1,4-dioxane, dimethyl sulfoxide,ethylene glycol, methanol, chloroform, hexane, acetone, N, N′-dimethylformamide, ethanol, isopropyl alcohol, butyl alcohol, pentyl alcohol,tetrahydrofuran, xylene, mesitylene, or any combinations thereof. Whenthe self-assembled gels include a solvent, the gels can include between0.01 and 18 (e.g., between 0.05 and 18, between 0.01 and 15, between0.05 and 15, between 0.1 and 15, between 0.5 and 15, between one and 15,or between one and 10) weight/volume percent of the generally recognizedas safe gelator in the solvent. For example, the gels can include from0.5 to 25 percent by weight of the GRAS first gelator and 0.5 to 25percent by weight of the non-independent second gelator.

In some embodiments, the self-assembled gel compositions are lyophilizedto remove a solvent, such that the resulting gels form xerogels.Xerogels can be in a powder form, which can be useful for inhalation orfor formation into pills for oral administration. As xerogels aresolvent free, they can have improved shelf-life and can be relativelyeasily transported and stored. To lyophilize self-assembled gels, thegels can be frozen (e.g., at −80° C.) and vacuum-dried over a period oftime to provide xerogels.

In some embodiments, instead of or in addition to a GRAS first gelator,the self-assembled gel compositions can be formed of amphiphilic3-aminobenzamide derivatives including a molecular weight of 2,500 orless. The 3-aminobenzamide derivatives can form nanostructures having amaximum dimension of 2 nm or more (see, supra). The 3-aminobenzamidederivatives can further one or more agents encapsulated within thenanostructures or non-covalently bonded to the nanostructures.

In some embodiments, the amphiphilic 3-aminobenzamide derivatives have astructure of formula (I):

wherein

A is CR₁R₂ or O, wherein R₁ and R₂ are each independently H or halogen;

E is C₁₋₂ alkylene, C₁₋₂ haloalkyl, or absent;

D is selected from the group consisting of C₃₋₂₀ alkyl, C₂₋₆ alkenyl,aryl, C₃₋₂₀ cycloalkyl, wherein each are optionally substituted with 1,2, 3, or 4 groups selected from the group consisting of C₁₋₄ alkoxy,C₁₋₈ alkyl, halo, C₁₋₈ haloalkyl, and nitro.

In some embodiments, A is CH₂ or O.

In some embodiments, E is CH₂ or CH₂CH₂.

In some embodiments, D is phenyl, optionally substituted with 1, 2, 3,or 4 groups selected from nitro and C₁₋₄ alkoxy.

In some embodiments, D is phenyl, optionally substituted with 1, 2, 3,or 4 groups selected from nitro and methoxy.

In some embodiments, D is ethylenyl.

In some embodiments, A is O; E is absent; and D is C₃₋₂₀ cycloalkyl,optionally substituted with 1, 2, 3, or 4 C₁₋₈ alkyl.

In some embodiments, A is CH₂ or O; E is absent, and D is C₃₋₂₀ alkyl.

In some embodiments, the amphiphilic 3-aminobenzamide derivative has astructure of formula (I):

wherein

A is CH₂ or O;

E is CH₂, CH₂CH₂, or absent; and

D is selected from the group consisting of C₃₋₂₀ alkyl, C₃₋₂₀cycloalkyl, ethylenyl, and phenyl, each optionally substituted with 1,2, 3, or 4 groups selected from nitro, C₁₋₄ alkoxy, and C₁₋₈ alkyl.

In some embodiments, the amphiphilic 3-aminobenzamide derivatives caninclude:

Self-Assembled Gel Compositions for Delivering Agents

In some embodiments, the self-assembled gel compositions can include oneor more encapsulated agents. The agents can be hydrophobic, such theagents can be relatively non-polar and thus prefer neutral molecules andnon-polar solvents. In some embodiments, the agents can be hydrophilic.The agents can have a molecular weight of less than or equal to about500,000 Da. As an example, the agents can include a steroid, ananti-inflammatory agent, a chemotherapeutic, a polypeptide, a nucleicacid, a polynucleotide, a polyribonucleotide, an anti-pain agent, ananti-pyretic agent, an anti-depression agent, a vasodilator, avasoconstrictor, an immune-suppressant, a tissue regeneration promoter,a vitamin, a vitamin derivative, a dye, a sensor, and/or a smallinterfering RNA. In some embodiments, the agents can include a polymer,such as poly(ethylene glycol), poly(ethylene oxide), hyaluronic acid,chitosan, carboxy methylcellulose, poly(ethylene glycol) di-acrylate,and poly(glycerol-co-sebasate acrylate), and/or any derivative thereof.In some embodiments, the agents include triamcinolone acetonide,dexamethasone, ethambutol, iodomethacin, camptothecin, paclitaxel,temozolomide, carmustine, PARP-inhibitors, and/or any derivativethereof. The encapsulated agents can be embedded between the lamellae ofa self-assembled gel, or embedded within the hydrophobic groups of thegelators forming the self-assembled gel.

In some embodiments, the encapsulated PARP-inhibitors include NU1025,BSI-201, AZD-2281, ABT-888, AGO-14699, 4-hydroxyquinazoline,3-aminobenzamide, 1,5-isoquinolinediol, 4-amino-1,8-napthalimide,O⁶-benzylguanine, and/or derivatives thereof.

In some embodiments, the agents can include insulin, an anticoagulant, ablood thinner, an antioxidant, a sleep medication, an enzyme inhibitor,a GPCR agonist or antagonists, a vaccine, an inhibitory ribonucleic acid(RNAi), a protein, a peptide, an enzyme a nutrition supplement, anantibody, and/or an aptamers. In some embodiments, the agents canpromote cell migration, proliferation, matrix production, celldifferentiation, transendothelial migration, transdifferentiation,re-programming, and/or anti-apoptosis. In certain embodiments, theagents can alter metabolism.

Methods of Making the Self-Assembled Gel Compositions

Generally, to form a self-assembled gel composition, a solvent, agelator, and optionally an agent to be encapsulated are added to acontainer to form a mixture. In some embodiments, the mixture caninclude one or more solvents, one or more gelators (e.g., GRASgelators), and/or one or more agents to be encapsulated. The mixture canbe heated and/or sonicated and/or placed in a bath to completelydissolve the gelator to form a homogeneous solution, and the solution isthen cooled and/or rested in an undisturbed location. The solution cantransition into a viscous gel after a given time period. Gelation isdeemed complete when no gravitational flow is observed upon inversion ofthe container. To remove an unencapsulated agent from the gels, the gelscan be repeatedly vortexed in a solvent that can dissolve the agent butthat does not interact with the gels. The supernatant solution can beremoved to extract any unencapsulated agent.

When the self-assembled gel compositions do not include a solvent, agelator (e.g., a GRAS or a non-GRAS gelator) can be combined with aliquid amphiphile (e.g., a non-independent vitamin-derived liquidamphiphile) to form a mixture. The mixture can include one or moregelators and one or more liquid amphiphiles. The mixture is thenheated/sonicated/placed in a bath to form a homogenous solution. Theresulting solution is then allowed to cool and/or rest in an undisturbedlocation. The solution can transition into a viscous gel after a giventime period.

In some embodiments, one or more gelators and optionally an agent to beencapsulated can be combined in the absence of a solvent to form amixture. The mixture is then heated/sonicated/placed in a bath to form ahomogenous solution. The resulting solution is then allowed to cooland/or rest in an undisturbed location. The solution can transition intoa viscous gel after a given time period.

In some embodiments, to encapsulate an agent, a melted gel including oneor more gelator and one or more solvents can be added to a solid agent,to an agent dissolved a the same one or more solvents, or to an agentdissolved or suspended in a gel-compatible solvent.

In some embodiments, the heating temperatures can be from 40 (e.g., from50, from 60, from 70, from 80, from 90, or from 100) to 110 (e.g., to100, to 90, to 80, to 70, to 60, or to 50° C. The mixtures can be heatedand/or sonicated and/or placed in a bath for a duration of from one(e.g., from five, from 10, from 15, from 20, or from 25) to 30 (to 25,to 20, to 15, to 10, or to five) minutes. The solutions can be cooled toa temperature of from 4 (e.g., from 10, from 20, or from 25) to 37(e.g., to 25, to 20, or to 10° C. and/or rested for a duration of from15 minutes (e.g., from 30 minutes, from 45 minutes) to one hour (e.g.,to 45 minutes, to 30 minutes).

As an example, 0.01-10 wt % of a GRAS first gelator and 0.01-10 wt % ofGRAS second gelator can be dissolved in dissolved in 1-150 μl of watermiscible organic solvent, optionally, 50-199 μl of either water orphosphate buffer saline (PBS) can be added to the mixture. Heating(40-110° C.) and/or sonication and/or placing in a bath for 1-30 minfollowed by cooling (4-37° C.) can occur to form assembled gels. In someembodiments, instead of GRAS gelators, one or more amphiphilic vitaminderivatives (e.g., a vitamin C ester, a vitamin A ester, a vitamin Eester) can be used.

As another example, 0.01-10 wt % of a GRAS first gelator, 0.01-10 wt %of a GRAS second gelator, and 0.01-8 wt % an agent of interest can bedissolved in 1-150 μl of water miscible organic solvent, subsequently50-199 μl of either water or phosphate buffer saline (PBS) was added.Heating (40-110° C.) and/or sonication and/or placing in a bath for 1-30min followed by cooling (4-37° C.) GRAS agent can occur to formassembled gels including an encapsulated agent. In some embodiments, theGRAS gelators can be dissolved in 1-150 μl of water miscible organicsolvent, and an agent of interest (e.g., 0.1-5 wt %, 0.01-8 wt %) inwater or PBS can be added to the GRAS gelator solution. In someembodiments, instead of GRAS gelators, one or more amphiphilic vitaminderivatives (e.g., a vitamin C ester, a vitamin A ester, a vitamin Eester) can be used.

When one gelator is a liquid, as an example, 0.01-15 wt % of a solidGRAS gelator and optionally 0.1-10 wt % of an agent of interest can beadded to a liquid GRAS gelator, or the gelators and the agent ofinterest can be dissolved in an water miscible or immiscible organicsolvent. In some embodiments, instead of GRAS gelators, one or moreamphiphilic vitamin derivatives (e.g., a vitamin C ester, a vitamin Aester, a vitamin E ester) can be used.

As another example, 0.01-15 wt % of an amphiphilic GRAS-agent and0.01-10 wt % of phospholipid (either cationic, or anionic, orzwitterionic) and 0.01-8 wt % of an agent of interest can be dissolvedin an organic (water miscible or immiscible) solvent by heating (40-110°C.) and/or sonication and/or placing in a bath for 1-30 min, followed bycooling to lower temperature (4-37° C.) to form self-assembledorganogels.

In some embodiments, 0.01-15 wt % of an amphiphilic GRAS gelator and0.01-10 wt % of polymer (either cationic, anionic or zwitterionic orneutral) can be dissolved in 1-150 μl of a water miscible solvent,subsequently 50-199 μl of either water or phosphate buffer saline (PBS)was added to form a mixture. Heating (40-110° C.) and/or sonicationand/or placing in a bath the mixture for 1-30 min, followed by coolingto lower temperature (4-37° C.) can provide self-assembled hydrogels.The mixture can optionally include 0.01-8 wt % of an agent of interest,dissolved in the water-miscible solvent, in the water, or PBS. In someembodiments, instead of a water miscible solvent, water, and/or PBS, theamphiphilic GRAS gelator, polymer, and/or agent of interest can bedissolved in a water miscible or immiscible organic solvent.

In some embodiments, self-assembled fibers are isolated through repeatedcycles of centrifugation (2000-25000 rpm for 2-15 min) and PBS washings,to provide water dispersible self-assembled fibers with varying overallcharge of the fibers.

Characteristics of Self-Assembled Gel Compositions, Uses, and Methods ofDelivery

The self-assembled gel compositions can be lubricious, such that whenthe gel compositions are administered to a surface, decreased wear iscaused to the surface by a given friction-inducing object in a givenamount of time. The self-assembled gel compositions can have recoverablerheological properties. For example, they can have an elastic modulus offrom 10 (e.g., from 100, from 1,000, from 2,500, from 5,000, or from7,500) to 10,000 (e.g., to 7,500, to 5,000, to 2,500, to 1,000, or to100) pascals and a viscous modulus of from 10 (e.g., from 100, from1,000, from 2,500, from 5,000, or from 7,500) to 10,000 (to 7,500, to5,000, to 2,500, to 1,000, or to 100) pascals.

When administered to a biological system, the gel compositions can becontrollably disassembled, for example, upon exposure to hydrolytic,enzymatic degradation conditions, or an external stimulus. Gel assemblycan include cleavage of a labile linkage in an amphiphilic gelator, suchas an ester, amide, anhydride, carbamate, phosphate-based linkages(e.g., phosphodiester), disulfide (—S—S—), acid-cleavable groups such as—OC(O)—, —C(O)O—, or —C═NN— that can be present between a hydrophobicand hydrophilic group within the gelator. Examples of labile linkagesare also described, for example, in PCT publication WO2010/033726,herein incorporated by reference in its entirety.

In some embodiments, encapsulated agents can be controllably releasedfrom the gel compositions upon gel disassembly. For example,encapsulated agents can be gradually released over a period of time(e.g., a day, a week, a month, six months, or a year). Depending on theparameters, the release can be delayed from minutes to days to months oreven years, for example, when gel compositions are administered underphysiological conditions (a pH of about 7.4 and a temperature of about37° C.). For example, the sustained release can be controlled by theconcentration of an enzyme and/or a temperature. For instance, sustainedrelease can be accelerated via high enzyme concentration. In someembodiments, the sustained release is delivered without a burst release,or with only a minimal burst release.

The stimuli can be found in biological systems. For example, in certainembodiments, gel compositions can be disassembled under biologicalconditions, e.g., conditions present in the blood or serum, orconditions present inside or outside the cell, tissue or organ. The gelcompositions can be disassembled only under conditions present in adisease state of a cell, tissue or organ, e.g., inflammation, thusallowing for release of an agent at targeted tissue and/or organ. Forexample, the gel compositions can include degradable linkages that arecleavable upon contact with an enzyme and/or through hydrolysis, such asester, amide, anhydride, and carbamate linkages. In some embodiments,phosphate-based linkages can be cleaved by phosphatases. In someembodiments, labile linkages are redox cleavable and are cleaved uponreduction or oxidation (e.g., —S—S—). In some embodiments, degradablelinkages are susceptible to temperature, for example cleavable at hightemperature, e.g., cleavable in the temperature range of 37-100° C.,40-100° C., 45-100° C., 50-100° C., 60-100° C., 70-100° C. In someembodiments, degradable linkages can be cleaved at physiologicaltemperatures (e.g., from 36 to 40° C., about 36° C., about 37° C., about38° C., about 39° C., about 40° C.). For example, linkages can becleaved by an increase in temperature. This can allow use of lowerdosages, because agents are only released at the required site. Anotherbenefit is lowering of toxicity to other organs and tissues. In certainembodiments, stimuli can be ultrasound, temperature, pH, metal ions,light, electrical stimuli, electromagnetic stimuli, and combinationsthereof.

When the self-assembled gel compositions include amphiphilic3-aminobenzamide derivatives, the gel compositions can includeencapsulated agents such as those discussed herein, and/orchemotherapeutic agents including temozolomide, carmustine,camptothecin, and/or paclitaxel. The amphiphilic 3-aminobenzamidederivatives, which are poly(ADP-ribose) (“PARP”) inhibitors, can enhance(e.g., potentiate) the efficacy of encapsulated agents. For example,amphiphilic 3-amino benzamides can act in synergy with encapsulatedagents by acting via complementary pathways in a biological system. Theself-assembled gel compositions can also be internalized by cells—suchthat biologically active gelators (e.g., amphiphilic 3-aminobenzamide)can be released together with encapsulated agents at the same locationwithin a biological system. Gelators and encapsulated agents that canact synergistically can include, for example, self-assembled gelcompositions including vitamin C derivative gelators (e.g., ascorbylalkanoate to increase iron absorption) and aloe (to increase absorptionof vitamin C and E) which together can increase vitamin uptake;self-assembled gel compositions including PARP inhibitor gelators (e.g.,amphiphilic 3-aminobenzamide) and cisplatin and/or BMS-536924 whichtogether can block cellular repair pathways; self-assembled gelcompositions including non-independent vitamin A derived gelators (e.g.,retinyl acetate, retinyl palmitate) and interferons can provideheightened immune response; self-assembled gel compositions including avitamin C derivative gelators (e.g., ascorbyl alkanoate) and vitamin K3can increase the death of cancer cells; self-assembled gel compositionsincluding non-independent vitamin E derived gelators (e.g., alphatocopherol acetate) and vitamin D can promote remyelination.

The self-assembled gel compositions, which can optionally includeencapsulated agents, can be used for treatment of a variety ofconditions, such as proteolytic diseases, including inflammatorydisease. In some embodiments, the gel compositions can be used aslubricants or viscosupplements to damaged joints. The gel compositionscan restore lubricant properties of synovial fluid ofarthritis/pathological joints. In some embodiments, the gel compositionscan be used for replacement of fluids such as synovial fluid, aqueoushumor, and/or vitreous humor. When gel compositions include encapsulatedinsulin, the gel compositions can be used for the treatment of diabetes.

In some embodiments, the self-assembled gel compositions can be used forcellular applications including cell delivery. The gel compositions canbe used for of delivery osteogenic agents to promote osteogenesis, aspart of an oral rinse to target the oral cavity, applied to the skin torelease agents for cosmetic or therapeutic purposes, used to treatulcers including mucosal and skin, used to treat tumors (examples mayinclude brain, skin, head and neck, breast, prostate, liver, pancreas,lung, bone, and/or oral), used to treat acute and chronic kidneydisease, and/or applied to treat gum disease. The gel compositions canbe components in tooth paste, shampoo/conditioner, soap, shaving cream,hand cream, sanitizer, makeup, eye drops, razors, nasal spray, nailpolish, hair spray/gel, shoe polish, paint, detergent, fabric softener,water purification, plaster, toilet cleaner, food. In some embodiments,the gel compositions can be delivered to the surface of the scalp topromote hair growth. In some embodiments, the gel compositions can beused to deliver of nutrient supplements in high concentrations wherevitamins are provided by the GRAS gelator and/or from entrappedvitamins.

In some embodiments, the self-assembled gel compositions can be used forprotection of skin from sunburns and inflammation, and delivery ofantioxidants—where anti-oxidant properties are provided by the GRASgelator and/or from entrapped antioxidants. The gel compositions can beused in the treatment of back pain, carpal tunnel syndrome, diabeticretinopathy, ulcerative colitis, crohn's disease, tennis elbow, heartdisease, cardiovascular disease, and peripheral vascular disease. Thegel compositions can be useful, e.g., for improving safety, targetingefficiency, compliance and efficacy for indications benefiting fromsingle dose, prolonged action or tissue-specific formulations. Exemplaryindications include, but are not limited to, allergy (e.g. contactdermatitis), arthritis, asthma, cancer, cardiovascular disease, diabeticulcers, eczema, infections, inflammation, muscuscitis, periodontaldisease, psoriasis, respiratory pathway diseases (e.g., tuberculosis),vascular occlusion, pain, graft versus host diseases, canker sores,mucositis, bacterial conditions, viral conditions.

In some embodiments, the self-assembled gel compositions can includestimulation of pathways to promote formation of extracellular matrix(i.e. gel composition induces formation of collagen that may findutility in cosmetic applications, or promotes formulation of new tissuessuch as muscle or other connective tissues).

It is possible to administer the gel compositions through various knowndelivery techniques, including injection and implantation. Injection andimplantation are particularly feasible in view of the ability of thegelator to form in situ (i.e. in situ self-assembly). Injecting orimplanting the gel compositions into a joint together with thesustained-release properties enables the gel compositions to provide along-term release of an encapsulated agent over a period of time. Thisis particularly suitable in instances where enzymes that are present ina joint are naturally released upon inflammation of the joint. When thejoint becomes inflamed and releases the enzyme, the enzyme, in turn,disassembles gel compositions, which releases the anti-inflammatorydrugs. After the anti-inflammatory drug is released, the enzymeconcentration decreases. The gel compositions that are not cleavedremain stable until another inflammatory stimulus. This phenomenon canbe referred to as “on-demand release,” where the level of inflammationregulates the amount and timing of an agent release. In someembodiments, the gel compositions can be useful to release therapeuticagents that correlate with different stages of tissue regeneration.

Application may be through systemic infusion, injection,transplantation, inhalation, or topical application including to themucosa, oral, buccal, nasal, intestinal, vaginal, rectal and skin. Thegel compositions can be spatially targeted when administered to abiological system. For example, the gel compositions can be locallydelivered via implants or injections, or the gel compositions can bysystemically delivered. The active transfer of amphiphiles through atissue can be enhanced/achieved by the action of electrical or otherforms of energy. These may include iontophoresis; sonophoresis andelectroporation. The gel compositions can be amenable to inner ear drugdelivery, oral drug delivery, ophthalmologic application, andincorporation within chewing gum for controlled release of agentsincluding flavoring agents, vitamins, or nutraceuticals. For example,the gel compositions can be in xerogel form and can be incorporated intoa lozenge or chewing gum for controlled release of flavoring agents,vitamins, or nutraceuticals.

The self-assembled gel compositions can be used in transdermal delivery(e.g., transdermal patches, permeabilization), and combined with otherexternal devices which can be applied on the skin. The gel compositionscan be used for intranasal delivery of drugs, in various cosmeticapplications including bulking agents or for applications whereproduction of extracellular matrix such as collagen is desired. Forexample, the gel compositions can be in xerogel form and administered toan intranasal cavity by inhalation. In some embodiments, the gelcompositions can be used for delivering drugs into the gut andinner-lumen of vessels through endoscopic application (endoluminalapplications), which can offer advantage over trans dermal patches thatcan induce inflammation or cause skin irritation.

The self-assembled gel compositions can be used with Natural OrificeTransluminal Endoscopic Surgery (“NOTES”) to localize drug deliverydevices within or between specific internal tissues. In someembodiments, the gel compositions can be delivered to a tumor forsustained delivery of chemotherapeutics, or can be delivered to a siteof healthy tissue following cancer resection to decrease the chances ofrecurrence. For example, gel compositions including a PARP inhibitor anda chemotherapeutic agent can be delivered by injection or byimplantation at a brain cancer site for sustained anticancer therapy, byblocking one or more cellular repair mechanisms.

In some embodiments, the gelators can be applied to a biological systemand self-assembly can occur in situ. For example, the gel compositionsdescribed herein may be applied to the surface of bone and the gel canbe assembled within the pores of the bone. For example, heated gelcompositions can be injected in solution form to a bone site, which canthen cool to physiological temperatures to assemble into gel forms.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1

The gelation ability of vitamin precursors in combination with othervitamin precursors or GRAS agents has been investigated. Representativeresults are presented below.

TABLE 1-1 1:1 retinyl acetate (vitamin A precursor) in ascorbylpalmitate (vitamin C precursor) Precipitation/ Wt % retinyl Wt %ascorbyl Gelation/ acetate palmitate Solvent Soluble (P/G/S) 6% 6% DMSOin water No gel 70% 6% 6% DMSO in water G 30% 6% 6% Ethanol in water G30% 6% 6% DMF in water G 30%

TABLE 1-2 1:2 Retinyl Acetate (vitamin A precursor) in Ascorbylpalmitate (vitamin C precursor) Precipitation/ Wt % retinyl Wt %ascorbyl Gelation/ acetate palmitate Solvent Soluble (P/G/S) 3% 6% DMSOin water G 30% 3% 6% Ethanol in water G 30% 3% 6% DMF in water G 30%

TABLE 1-3 1:1 Alpha-tocopherol Acetate (vitamin E precursor) in Ascorbylpalmitate Precipitation/ Wt % tocopherol Wt % ascorbyl Gelation/ acetatepalmitate Solvent Soluble (P/G/S) 4% 4% DMSO in water No gel 30% 4% 4%DMF in water G 30% 4% 4% Acetone in water G 30% 4% 4% Ethanol in water G30%

TABLE 1-4 25:1 Alpha-tocopherol Acetate (vitamin E precursor) inAscorbyl palmitate (vitamin C precursor) Alpha tocopherol Ascorbylpalmitate Solvent G/P/S 0.2 g 8 mg DMSO - 80 μl S H₂O - 20 μl Emulsion0.2 g 8 mg EtOH - 80 μl S H₂O - 20 μl G 0.2 g 8 mg CHCl₃ - 80 μl G 0.2 g8 mg Toluene - 80 μl S

TABLE 1-5 Using Alpha tocopherol Acetate and Retinyl Palmitate as thesolvent Solvent/second non- GRAS gelator independent gelator ResultAscorbyl palmitate Alpha tocopherol acetate 20 mg (9%)   0.2 g Insoluble20 mg (6.25%) 0.3 g Insoluble Ascorbyl palmitate Retinyl palmitate 3 mg(1.5%) 0.2 g Precipitate Sorbitan monostearate Retinyl palmitate 5 mg(2.5%) 0.2 g Soluble 6 mg (3%)  0.2 g Soluble Triglycerol monostearateAlpha tocopherol acetate 3 mg (1.5%) 0.2 g Soluble 9 mg (4.5%) 0.2 g Gel

Example 2

We have focused our attention on the development of enzyme-responsiveself-assembled nano/microfibrous hydrogels that can easily be injectedinto the articular space, yet are much larger than free drug, whichshould increase residence time by preventing rapid clearance by thelymphatic system. The inherent nanometer-scale features of thisself-assembled noncrosslinked hydrogel maximize the interaction withspecific enzymes for rapid disassembly and drug release. Gels made fromascorbyl palmitate (Asc-Pal) can encapsulate model agents, withstandshear forces that may be experienced in dynamic environments such asjoints, remain stable following injection into healthy joints of mice,and can disassemble in vitro to release encapsulated agents in responseto synovial fluid from arthritic patients. A graphical representation ofan agent encapsulation by a self-assembling gel, and the subsequentdisassembly and agent release process is shown, for example, in FIG. 1.

Ascorbyl palmitate (“Asc-Pal”) and matrix metalloproteinases werepurchased from Sigma Aldrich (St. Louis, Mo.). The Novozyme 435 (lipaseB from Candida antarctica) and Lipolase 1001 were obtained fromNovozymes through Brenntag North America. 1,10-Dioctadecyl-3,3,30,30-tetramethylindodicarbocyanine,chlorobenzenesulfonate salt (DiD) dye was purchased from Invitrogen.

Preparation of Gels

Typically, solvents (0.2 mL) were added to a glass scintillation vialwith the gelator (0.5-5 wt/vol %) and sealed with a screw cap. The vialwas heated to about 60-80° C. until the gelator was completelydissolved. The vial was placed on a stable surface and allowed to coolto room temperature. Typically after 15-45 min, the solution wastransitioned into a viscous gel. Gelation was considered to haveoccurred when no gravitational flow was observed upon inversion of theglass vial, and resulted hydrogels are injectable. Asc-Pal formed gelsin water, benzene, toluene, carbon tetrachloride, and acetonitrile;while a precipitate formed in dimethylformamide and dimethylsulfoxide.Asc-Pal was soluble in chloroform and methanol.

The morphologies of the self-assembled hydrogels were examined using SEMand fluorescence polarizable optical microscopy. Investigation of thehydrogels formed from Asc-Pal with SEM showed that hydrogels formfibrous structures with fiber thicknesses of 20-300 nm and fiber lengthsof several microns. The anisotropic nature of intermolecularinteractions between amphiphile molecules is supported by the highaspect ratios of the gel fibers. Dye-encapsulating fibers were rinsedwith excess PBS to remove unencapsulated dye, and subsequentfluorescence microscope images of the fibers indicated that the dye wasencapsulated within the fibers.

Enzyme-Responsive Fiber Disassembly and Release of Dye

We demonstrate the encapsulation of a model dye in Asc-Pal hydrogel,which upon enzyme-mediated gel degradation releases the encapsulated dyeat model physiological conditions in a controlled manner. Specificenzymes are significantly upregulated within arthritic joints, and theirexpression and concentration correlate with the degree of synovialinflammation. Thus, we have tested the ability of self-assembled gelfibers to release an encapsulated payload in response to the enzymesthat are expressed within arthritic joints.

DiD-encapsulating gel fibers were dispersed within PBS and incubated at37″C with either lipase (esterase or MMP-2, or MMP-9 enzyme (100 ng/mL).At regular intervals, aliquots of samples were collected, and release ofthe dye was quantified using absorption spectroscopy. Plottingcumulative release of the dye (%) versus time (FIG. 2A) revealed thatlipase and MMPs trigger fiber disassembly to release the encapsulateddye, whereas gels in PBS controls remained stable and did not releasesignificant amounts of dye. Additionally, through thin-layerchromatography, we identified the presence of ascorbic acid and palmiticacid (confirmed by comparing Rf values by cospotting with authenticsamples of ascorbic and palmitic acids) only in gel solutions thatcontained enzymes. We have shown that gels in PBS remain stable for atleast 3 months, indicating that the presence of enzymes is required forgel disassembly and the release of encapsulated agents. This resultconfirms the absence of loosely bound dye on the surface of the gelfibers.

In the absence of enzymes, mechanical agitation of the fibers throughrigorous vortexing did not induce the release of dye, indicating thatagents incorporated within the fibers remain stably entrapped.Importantly, in the present system, we did not observe burst release(FIGS. 2A-2B). To investigate the potential for on-demand disassembly,following a 4-day incubation with enzyme (MMP-2, MMP-9, or lipase)containing media that triggered disassembly of fibers, media werereplaced with PBS, which halted the disassembly of fibers and therelease of dye. After a subsequent 7-day incubation with PBS, enzymeswere added to the suspended fibers, triggering disassembly and therelease of the encapsulated dye (FIG. 2B). These results clearly suggestthat Asc-Pal self-assembled fibers respond to proteolytic enzymes thatare present within arthritic joints and release encapsulated agents inan on-demand manner.

Arthritic Synovial Fluid Induces Fiber Disassembly

DiD-encapsulating fibers were incubated in arthritic synovial fluid at37° C., and the release of dye was quantified over a period of 15 days.Plotting cumulative release of the dye (%) versus time (FIG. 3A)revealed that synovial fluid triggers fiber disassembly leading to therelease of the dye. To determine whether proteases that are present inarthritic joints were responsible for fiber disassembly, we preparedlysates from arthritic joints of mice in the presence and absence ofprotease inhibitors. Incubation of self-assembled gel fibers with theselysates was used to help reveal the role of arthritis-associatedproteases. The presence of protease inhibitors significantly reducedfiber disassembly and dye release, thus demonstrating that the presenceof enzymes was critical for promoting the release of agents from gelsformed from Asc-Pal (FIG. 3B.).

Fiber Stability in Joints Under Nonarthritic Conditions

To investigate the stability of fibers in the absence of inflammation,fibers were injected into the joints of healthy mice using a small-bore(27 gauge) needle. Eight weeks post-implantation, the ankles of micewere sectioned and imaged with optical and fluorescence microscopy toobserve the presence of fibers. Images of tissue sections revealed thatDiD-encapsulating fibers were present, suggesting the potential forlong-term hydrolytic stability of the fibers in vivo.

Reversible Self-Assembly of Fibers

Materials that are injected into the joint space experience cyclicalmechanical forces during ambulation; thus, it is important thatmaterials that are injected into the joint can withstand these forcesand retain their characteristic material properties such as mechanicalstrength and morphology.

To investigate the impact of relevant mechanical forces on the Asc-Palfibers, we subjected gel nanofibers to cyclical shear forces andexamined their resulting rheological properties using a rheometerequipped with a parallel-plate geometry (FIG. 4A). The elastic/storagemodulus G′ was independent of frequency and was much higher than theviscous modulus G″ over the frequency range (0-12 rad/s) examined (FIG.4A). This type of response is typical of gels, as it shows that thesample does not change its properties or “relax” over long time scales.The value of G′ is a measure of the gel stiffness, and its value here(>1000 Pa) indicates a gel of slightly higher strength than collagenplatelet gels. The mechanical properties and strength of these gels arecomparable with earlier reported self assembled peptide gels that arebeing examined as possible injectable joint lubricants for the treatmentof osteoarthritis.

Frequency sweeps conducted before/after multiple cycles (1, 20, and 40)of a high shear stress were used to measure G′ (storage modulus).Interestingly, no significant differences were observed after 40 cycles(FIG. 4B), indicating that the gel fibers retain their mechanicalstrength.

These results indicate that self-assembled fibers made of Asc-Pal havethe potential to retain their morphology and mechanical properties evenunder the dynamic forces that may be experienced during ambulation.

Example 3

We set out to design an Abz-derived amphiphile that would encapsulatetemozolomide, and release it only upon contact with enzymes closelyassociated with the presence of glioma cells, namely, the family ofmatrix metallopeptidases (MMPs) commonly found at sites of inflammation.By adding a hydrophobic group to the water-soluble Abz through acarbamate linkage, a hydrolytically stable, enzyme cleavable amphiphilecould be synthesized (FIG. 5). This amphiphile could then be made toundergo self-assembly either alone or in the presence of temozolomide,in which case the chemotherapeutic agent would be encapsulated withinthe gel. Such a temozolomide-encapsulating Abz-derived gel would then beinjected into the tumor cavity post-resection, providing an on-demandreservoir of chemotherapeutic agent in conjunction with free Abz, bothto be released upon contact with glioma cells.

A variety of Abz-derived amphiphiles were synthesized (Scheme 3-1).Gelation conditions were explored for the set of amphiphiles, with theexception of Abz-PNP, which was found to decompose spontaneously. Ingeneral, mixtures of polar organic solvents with water gave the bestgelation results, with only two pure organogels forming (Abz-Alloc inglycerol, and Abz-Chol in 1,4-dioxane). Solvent systems that were foundto promote gelation were further explored with respect to concentrationand ratio of solvents. The four amphiphiles had widely differentsolubility and gelation properties, with each amphiphile forming atleast two gels.

Gels formed from Abz-ONB and Abz-Chol both presented as fibrousstructures under bright field microscopy, suggesting the formation ofself-assembled fibers. Gels of Abz-Alloc and Abz-Cbz, however, did notreveal any fibers, and instead appeared highly crystalline in nature,suggesting that these amphiphiles were not forming fibrous gels.

We tested the encapsulation abilities of Abz-ONB and Abz-Chol byincluding curcumin in the organic solvent used for gelation. Such gelswere washed with excess water and then observed under the FITC channelon the fluorescent microscope. The presence of curcumin could beobserved in the gels of both Abz-ONB and Abz-Chol, and was located onlywithin the fibers.

We next moved to testing the encapsulation of temozolomide. Abz-Cholsuccessfully encapsulated temozolomide. We sought to use milder gelationconditions while allowing for flexibility in drug loading. Severalmethods were attempted, (a) adding the melted gel to solid temozolomide,(b) temozolomide wetted with the gelation solvent, and (c) temozolomidewetted with water. All three systems yielded nearly equivalent loading.We chose method (a) for continued use, and we hypothesized that avoidingcontact between temozolomide and solvent for as long as possible wouldhelp to prevent degradation during formation. The weight-percent loadingbegan to plateau at approximately 25%, which corresponds to a 1:1 moleratio of temozolomide to Abz-Chol. A system including 6 mg oftemozolomide in 12 mg of Abz-Chol was chosen for further investigation.

We characterized both the native Abz-Chol gel and thetemozolomide-encapsulating gel through both bright field microscopy andscanning electron microscopy (SEM), and observed significantly differentmorphology. Under the light microscope, the native gel appearedsemi-crystalline, while the encapsulating gel appeared as fluffy clumps.Under the electron microscope, the native gel appeared as large needlesthat upon further inspection were made from the tight association ofmicro and nano scale fibers. The encapsulating gel appeared as a highlyfibrous interwoven network of micro and nano scale fibers, indicatingthat although the presence of temozolomide does not inhibit gelation, itdoes significantly change the ability of the formed fibers to aggregateinto semi-crystalline bundles.

We performed both stability and release studies on gels dispersed in theappropriate media (hereafter referred to as gel), and lyophilized gelsdispersed in the appropriate media (hereafter referred to as xerogel).We first investigated the stability of temozolomide in fibers dispersedin DPBS over the course of two days compared to a control of freetemozolomide in DPBS solution (FIG. 6). We achieved this by dissolvingaliquots of fibers in DMSO at specified time points and measuring thetemozolomide concentration inside by HPLC (Atlantis column). The datasuggest that temozolomide is protected from hydrolysis, and degrades ata significantly slower rate both in gel and xerogel than freetemozolomide. This is a very important indication, as an on-demand drugdelivery depot would need to stabilize the rapidly hydrolyzedtemozolomide until it is delivered.

We took unwashed gel and xerogel containing temozolomide, dispersed thefibers in DPBS, and followed the supernatant concentration oftemozolomide and AIC over the course of two weeks by HPLC (Atlantiscolumn). In both formulations we observed an expected initial burstassociated with the nonencapsulated temozolomide (FIG. 7); however, thiswas much greater in the case of the gel (57% vs. 31% for xerogel). Bothformulations were relatively stable over the first day; however, overthe course of the experiment the gel continued to release temozolomide,eventually releasing the full load, while the xerogel did not releasebeyond the expected burst.

We also evaluated the ex vitro release profile of the transwell-basedxerogel formulation. Transwells containing the air-dried in situ formedxerogels were incubated in DPBS in triplicate. At each time point themedia was removed, the volume measured, and the composition measured byHPLC, replacing with fresh media. In this manner cumulative release wasmeasured over the course of two weeks, the results of which arepresented in FIG. 8. We observed a significantly smaller burst releasecompared with previous release studies, followed by a steady release oftemozolomide, reaching approximately 60% after 15 days. The lower burstrelease was likely the result of less agitation in the context of a cellculture plate.

In order to further understand the mechanism of temozolomideencapsulation, and to probe the effects of enzyme-catalyzed degradation,we synthesized two additional Abz-derived amphiphiles (Scheme 3-2).Structures were chosen to be as similar as possible with the exceptionof one being a carbamate and the other an amide.

In sum, we have developed a series of novel 3-aminobenzamide-derivedamphiphiles, which can be made to self-assemble in a variety of organicand aqueous solvent systems to form micro and nano fibrous structures.Under appropriate conditions, such gels were shown to encapsulate eithercurcumin or temozolomide, without disrupting self-assembly. Encapsulatedtemozolomide was stabilized against hydrolysis within the gel fibers,and the results of several release studies showed controlled release ofdrug under physiological conditions from several formulations. Such asystem has broad applicability in the development of new deliverysystems for several forms of brain cancer. Additionally, a new systemwas developed to allow for the delivery of hydrophobic xerogels to cellsin culture through the use of transwell inserts, which can be used infuture cell-based assays.

Materials and Methods

Synthesis

Abz-ONB:

To a solution of 3-aminobenzamide (223 mg, 1.6 mmol) in 1/11,4-dioxane/10% AcOH(aq) (26 mL total) was added4,5-Dimethoxy-2-nitrobenzyl chloroformate (475 mg, 1.72 mmol) withstirring at r.t., producing a yellow suspension. After 23 hours thesuspension was filtered through qualitative filter paper, washing withwater. The collected yellow solid was dried in vacuo to give Abz-ONB(553 mg, 1.47 mmol, 90% yield). NMR in CDCl₃ with CD₃OD (500 MHz).

Abz-Alloc:

To a solution of 3-aminobenzamide (681 mg, 5 mmol) in 1/11,4-dioxane/10% AcOH(aq) (80 mL total) was added allyl chloroformate(0.56 mL, 5.25 mmol) with stirring at r.t. After 17 hours the clearsolution was basified to pH 10 with 2 M NaOH(aq) and diluted with 60 mLof water to form a cloudy suspension that was stirred at r.t. for 2hours. The mixture was diluted to a total volume of 250 mL with waterand filtered through qualitative filter paper and dried in vacuo to giveAbz-Alloc as an off-white solid (284 mg, 1.3 mmol, 26% yield). NMR inCDCl₃ with CD₃OD (300 MHz).

Abz-Chol:

To a flask containing 3-aminobenzamide (1.36 g, 10 mmol) and cholesterolchloroformate (4.72 g, 10.5 mmol) were added 1,4-dioxane (160 mL) andglacial acetic acid (8 mL) with vigorous stirring, generating a milkysuspension that turned pale pink. The reaction was stirred at r.t. for23 hours, at which point the suspension was basified to pH 10 with 2 MNaOH(aq), and diluted to a total volume of 600 mL with H₂O. Theresultant off-white suspension was filtered through qualitative paper,washing with water to give a white solid and a yellow filtrate. Thedried solid was washed with hexanes at 100 mg/mL 8 times, centrifugingto remove wash solvent. The resultant solvent was dried in vacuo to giveAbz-Chol as a white solid (2.88 g, 5.2 mmol, 52% yield). NMR in DMSO-d6(500 MHz).

Abz-Cbz:

To a solution of 3-aminobenzamide (681 mg, 5 mmol) in 1/11,4-dioxane/10% AcOH(aq) (80 mL total) was added benzyl chloroformate(745 μL, 5.25 mmol) in one portion with stirring. The resultant paleyellow solution was stirred at r.t. overnight, at which point it wasbasified to pH 11 with 2 M NaOH(aq), resulting in the formation of anoff-white precipitate. The suspension was filtered through qualitativepaper, washing with water and drying on filter to give Abz-Cbz (1.08 g,4.0 mmol, 80% yield). NMR in CDCl₃ with CD₃OD (300 MHz).

Abz-O-10:

To a solution of 3-aminobenzamide (1.36 g, 10 mmol) in 1,4-dioxane (20mL) and 10% AcOH(aq) (140 mL) was added decyl chloroformate (2.42 mL,10.5 mmol) in one portion with stirring. The resultant cloudy emulsionwas stirred at r.t. for 4 days, at which point the resultant orangesuspension was basified to pH 10 with 2 M NaOH(aq), and filtered throughqualitative paper, washing with water. The collected solid was dried invacuo to give Abz-O-10 as a tan solid. NMR in DMSO-d6 (500 MHz).

Abz-C-11.

To a solution of 3-aminobenzamide (681 mg, 5 mmol) in 1,4-dioxane (20mL) and 10% AcOH(aq) (60 mL) was added dodecanoyl chloride (1.21 mL,5.25 mmol) in one portion with stirring. The resultant off-whitesuspension was stirred at r.t. for 3 days, at which point the suspensionwas basified to pH 9 with 2 M NaOH and filtered through qualitativepaper, washing with water. The collected solid was dried in vacuo togive Abz-C-11 as a white solid. NMR in DMSO-d6 (500 MHz).

Gelation.

To a vial containing solid amphiphile was added the appropriate solvent.The vial was sealed and heated over a heat gun (˜110° C. max) untilamphiphile fully dissolved. The vial was allowed to cool undisturbed atroom temperature, during which time gel formed. For all characterizationgels were allowed to form for at least 45 minutes.

Encapsulation of Curcumin in Abz-Chol.

To a vial containing Abz-Chol (6 mg) was added curcumin in 1,4-dioxane(90 μL of 10 mg/mL). The vial was sealed and heated until fulldissolution, at which point H₂O (10 μL) was added, causing gelation. Thevial was again heated to full dissolution and allowed to cool, forming ayellow gel. The gel was mechanically broken and washed with H₂O(Millipore, 3×1 mL), centrifuging to remove washings. The resultantfibers were observed under bright field and fluorescence microscopy.

Encapsulation Efficiency and Loading.

To each vial containing Abz-Chol (12 mg) were added 1,4-dioxane (180 μL)and H₂O (20 Each vial was sealed and heated until full dissolution, andallowed to gel for a minimum of 1 hour. The gels were again heated tomelting, quickly transferred to vials containing temozolomide (1, 3, 6,or 12 mg; each in triplicate), and allowed to gel for a minimum of 1hour. Each gel was broken with pH 5 H₃PO₄(aq) (200 μL), and transferredto a 1.5 mL Eppendorf tube with pH 5 H₃PO₄(aq) (2×400 μL). Thesuspension was vortexed, centrifuged, and the supernatant removed.Fibers were washed with pH 5 H₃PO₄(aq) (1×1 mL), centrifuging to removesupernatant, and the composition of the combined supernatants wasdetermined by HPLC (pondapak) after passing through a 0.2 μm Nylon orPTFE syringe filter.

Release and Stability

Preparation of all Gels.

To a vial containing Abz-Chol (12 mg) were added 1,4-dioxane (180 μL)and H₂O (20 μL). The vial was sealed and heated until full dissolution,and allowed to gel for a minimum of 1 hour. The gel was again heated tomelting, quickly transferred to a vial containing temozolomide (6 mg),and allowed to gel for a minimum of 1 hour. Gels were then used as-is(gel), or frozen at −80° C. for a minimum of 30 minutes and lyophilizedovernight (xerogel).

Release in DPBS.

6% wt/vol xerogels made on 200 μL scale, with 6 mg temozolomide loadedinto each. Vials containing full xerogels were each placed in 20 mLscintillation vials, and DPBS (18.0 mL) was added, completely fillingthe inner vial. Larger vials were sealed and vortexed until xerogelswere uniformly dispersed, and then stored in 37° C. incubator. At eachtime point vials were vortexed and 100 μL of suspension was removed,diluted 10×, and measured by HPLC (pondapak) following filtrationthrough 0.2 μm Nylon syringe filter. Removed volume (100 μL) from vialswas replaced at end of each time point.

Transwell Experiments

Transwell Insert for Cell Assay.

Three sets of gels were formed, loading 6 mg, 3 mg, and 1 mg oftemozolomide in 12 mg of Abz-Chol. In each case, gels were melted andadded to vials containing the appropriate amount of temozolomide. Thevials were shaken briefly and immediately 2 μL of the resultantsuspension was pipetted to the membrane of a transwell insert.Additionally, a control was prepared of Abz-Chol gel with notemozolomide. Each was performed in quadruplet. Transwell inserts wereair dried for 1 week prior to use in cell culture.

Transwell Release.

To a vial containing Abz-Chol (12 mg) were added 1,4-dioxane (180 μL)and H₂O (20 μL). The vial was sealed and heated until full dissolution,and allowed to gel for a minimum of 1 hour. The gel was again heated tomelting, quickly transferred to a vial containing temozolomide (6 mg),shaken to mix, and 4 μL aliquots added to the membrane of each of 624-well transwell inserts. Gels were allowed to dry open for 30 minutes,and then covered for 2 days. Inserts were added to 24-well plate andkept under DPBS or Lipolase (100 U/mL diluted 10× in DPBS) (1.5 mLinitially, 1.0 mL for subsequent time points) in 37° C. cell cultureincubator, each in triplicate. At each time point the media was removedand replaced with fresh media of the same variety. The volume of theremoved media was measured, and the composition measured by HPLC(pondapak) following filtration through 0.2 μM Nylon or PTFE syringefilter. Cumulative release was thus calculated.

Example 4

Camptothecin was encapsulated in a PARP inhibitor. Camptothecin is acytotoxic alkaloid which inhibits the DNA enzyme topoisomerase. DNAtopoisomerase helps in relieving the torsional strain in the DNA duringreplication. Camptothecin binds to the topoisomerase 1 nicked DNAcomplex and prevents relegation. Since the DNA is damaged the cellundergoes apoptosis. However the cells have an inherent mechanism torectify the process. An enzyme known as PARP gets activated when the DNAstrand is broken and recruits DNA repairing enzyme and repairs thebroken DNA. Camptothecin is sparingly soluble in water, easilyconvertible to inactive carboxylate form and undesirable systemic sideeffects. Hence an effective method for treatment of glioblastomas wouldbe to co-encapsulate both Camptothecin and PARP inhibitor and releasethe drug in a sustained manner for a long period of time.

We have investigated the self-assembled ability of sorbitan monostearate(SMS, GRAS-agent, Scheme 4-1) in a wide range of solvents, minimumgelation concentrations in various solvents, and their morphology usingelectron microscope. In addition, we have also investigated the abilityof SMS gels to encapsulate a chemotherapeutic agent, camptothecin (CPT)and different PARP-inhibitors. Encapsulation efficiency, loadingcapacity, stability of gels and detailed release kinetics in response tothe enzymes have been investigated. Detailed results have beensummarized in following sections.

ResultsGelation Ability of SMS.

Table 4-1 shows the versatile self-assembling ability of SMS in varioussolvents. Intriguingly, SMS has demonstrated appreciable ability toself-assemble in aqueous solution and also in polar aprotic (dimethylsulfoxide, dimethyl formamide) and protic solvents (ethylene glycol,polyethylene glycol) as well as non-polar solvents like isopropylpalmitate, hexanes and hexadecane (Table 4-1). These results clearlyindicate the versatile gelation ability of SMS amphiphile.

TABLE 4-1 Gelation ability of amphiphile SMS in a wide range ofsolvents. Solvent Sorbitane Monostearate Water Gel Dimethyl sulfoxide(DMSO) Gel Dimethyl formamide (DMF) Soluble Acetone PrecipitateAcetonitrile (CH₃CN) Precipitate Methanol Precipitate Ethanol SolubleIsopropanol Soluble Isobutanol Soluble Glycerol Gel Mono-ethylene glycolWeak Gel Polyethylene glycol dimethyl acrylate Increased ViscosityDioxane Soluble Tetrahydrofuran (THF) Soluble Isopropyl palmitate ClearGel Ethyl acetate Emulsion Chloroform Soluble n-hexane Gel n-hexadecaneGel Toluene Soluble Xylene Soluble Tetrachloro methane SolubleConcentration was around 5-9%

In addition, SMS has demonstrated different minimum gelationconcentrations (MGC) in different solvents (Table 4-2). MGC values ofSMS have varied from 3 to 8 wt/v %.

TABLE 4-2 Minimum gelation concentration of amphiphile SMS in gellingsolvents. Sorbitane Minimum Gelation Solvent Monostearate Concentration(Wt/V %) Water Gel 3% Dimethyl sulfoxide (DMSO) Gel 3% Ethylene glycolGel 5% Glycerol Gel 8% Isopropyl palmitate Gel 3% n-hexane Gel 4%n-hexadecane Gel 2%Encapsulation of Camptothecin.

Chemotherapeutic agent camptothecin (CPT) is a hydrophobic drug, whichhas very low water solubility. Thus, we investigated the encapsulationability in SMS gels. Results are summarized in Table 4-3. Interestingly,SMS gels have shown high loading ability, i.e., 4 and 6% (wt/v) SMS gelswere shown ˜30% (wt/wt) loading efficacy (Table 4-3).

TABLE 4-3 Camptothecin loading efficiency of self-assembled gels of SMSin DMSO Sorbitane Monostearate, Camptothecin starting Loading efficiency% (wt/v) concentration (mg) % (wt/wt) 4 4 32.28 6 4 30.02 8 4 18.25 12 412.67Morphology of Self-Assembled Gels of SMS.

We have characterized the morphology of self-assembled gels of SMS intheir native gel form as well as CPT encapsulated gels. DMSO gels of SMSand CPT-encapsulated gels both show similar morphology under scanningelectron microscopy.

Similarly, we have characterized the morphology of hydrogels (20% (v/v)DMSO in water) of CPT encapsulated SMS gels. Interestingly, at differentweight percent of SMS (e.g., 4%, 8% and 12% (wt/v)) these gels showedonly moderate differences in their morphology.

Controlled Release of Chemotherapeutic Agent.

Often drug delivery vehicles suffer from burst release and continuousrelease of cargo due to degradation of vehicles. Chemotherapeutic agentcamptothecin (CPT) has been encapsulated in SMS gels with higher loadingefficiency (30% (wt/wt). Release kinetic experiments reveal that CPTencapsulated SMS gels do not have burst release (FIG. 9). In the absenceof enzymes, these gels were stable and showed only moderate release ofCPT (˜15%) and reached plateau in ˜8-10 hr. On contrary, in the presenceof an esterase enzyme (lipase, 10,000 units) has showedenzyme-responsive release of CPT, which suggests that esterase enzymecan degrade the SMS gels in an on-demand manner to release theencapsulated drugs in a controlled manner (FIG. 9).

Cytotoxicity Studies of CPT Encapsulated SMS Self-Assembled Fibers.

Prior to in vivo studies, evaluation of efficacy of drug deliverysystems using appropriate cell lines is an essential step. Efficacy ofCPT loaded SMS self-assembled fibers were investigated using glioma cellline, G55 and control cell line fibroblast, NIH3T3. When CPT-loaded SMSself-assembled fibers incubated in culture with G55 cells, the IC₅₀ ofCPT-loaded SMS fibers was 15 nM, compared to 30 nM for CPT alone (FIG.10A). On contrary, when CPT-loaded SMS self-assembled fibers incubatedin culture with fibroblasts (NIH3T3), the IC₅₀ of CPT-loaded SMS fiberswas 62.5 nM, compared to 3.9 for CPT alone (FIG. 10B). Intriguingly,CPT-loaded SMS fibers decreased 2-fold of IC₅₀ for gliomas G55, whereasCPT-loaded SMS fibers increased 15-fold of IC₅₀ for fibroblasts. Wehypothesize that highly proliferative glioma G55 cells secretes highconcentrations of enzymes that degrade the self-assembled fibers torelease CPT efficiently, whereas free form of CPT degrades in the mediadue to its low half-life at physiological conditions. On contrary,fibroblast could not degrade the fibers to release CPT, thus IC₅₀ ofencapsulated CPT (protected from hydrolytic degradation) has increasedIC₅₀ (˜15-fold). These results indicate that proliferative glioma cellstriggers the drug release in an on-demand manner.

Encapsulation of Camptothecin and PARP-Inhibitor (AGO14699).

Chemotherapeutic agent camptothecin (CPT) is a hydrophobic drug, andPARP-inhibitor AGO14699 has moderate water solubility. Thus, both agentswere encapsulated in hydrogel fibers of SMS to evaluate theirsynergistic efficacy against glioblastoma (brain cancer) cell lines.Inherent gelation ability of SMS did not altered in the presence of CPTand AGO14699; gelation results are summarized in Table 4-4. CPT andAGO14699 have been efficiently loaded in SMS hydrogel fibers with ˜30and 15% (wt/wt), respectively (Table 4-5).

TABLE 4-4 Gelation ability of sorbitan monostearate in the presence ofcamptothecin (CPT) and PARP-inhibitor (AGO-14699). SorbitaneMonostearate + Solvent Camptothecin + PARP inhibitor Water Gel Dimethylsulfoxide (DMSO) Gel Isopropyl palmitate Gel Ethanol Soluble IsopropanolSoluble Isobutanol Soluble Ethylene glycol Gel Polyethylene glycol WeakGel Polyethylene glycol dimethyl acrylate Increased Viscosity

TABLE 4-5 Loading efficiency of camptothecin (CPT) and PARP-inhibitor(AGO-14699) in sorbitan monostearate DMSO gels. Loading efficiency %(wt/wt) PARP-inhibitor (AGO14699), Camptothecin (CPT) 15.10 30.02Cytotoxicity Studies of CPT and AGO14699 (PARP-Inhibitor) EncapsulatedSMS Self-Assembled Hydrogels

Efficacy of CPT and PARP-inhibitor loaded SMS self-assembled hydrogelswere investigated using glioma cell lines G55 and U87. When CPT-PARPinhibitor loaded SMS hydrogels incubated in culture with G55 cells, theIC₅₀ of CPT-loaded and PARP-inhibitor loaded SMS gels were 105 nM and1875 nM, compared to 36 nM for combination gel alone (FIG. 11A). Thus,3-fold enhanced efficacy of chemotherapeutic agent, CPT has beenobserved with co-local delivery of CPT and PARP-inhibitor throughself-assembled hydrogels. Interestingly, similar trend has been observedagainst U87 glioma cell line as well (FIG. 11B).

In summary, the SMS amphiphile has demonstrated an unprecedented abilityto form self-assembled gels in a wide range of solvents including polar,non-polar, protic and aprotic solvents. SMS gels can encapsulate CPTwith high loading efficiency, and stabilize the CPT to protect it fromhydrolytic degradation. CPT-loaded SMS fibers can release CPT in thepresence of ester enzymes in an on-demand manner. Cytotoxicity studiesare in agreement with in vitro release studies, CPT-loaded SMSself-assembled fibers are very effective against glioma cells G55.

Example 5

We have also investigated the ability of SMS gels to encapsulate ananti-inflammatory agent, triamcinolone acetonide (TA, Scheme 5-1).Loading efficiency, stability of gels, and release kinetics in responseto the presence of enzymes has been investigated.

Scheme 5-1.

Chemical structure of corticosteroid based anti-inflammatory agent,triamcinolone acetonide (TA).

Morphology of Self-Assembled Gels of Triamcinolone AcetonideEncapsulated Hydrogels.

Scanning electron microscope images revealed that TA encapsulated SMShydrogels (20% (v/v) DMSO in water) are highly porous and similar to SMSgels without TA.

Controlled Release of Triamcinolone Acetonide.

Corticosteroid TA has been encapsulated in SMS gels with higher loadingefficiency. Release kinetic experiments reveal that TA encapsulated SMSgels do not exhibit burst release (FIG. 12). In the absence of enzymes,these gels were stable and showed only moderate release of TA (˜20%)reaching a plateau within ˜24 hr. On the contrary, in the presence of anesterase enzyme (lipase, 10,000 units), gels exhibited enzyme-responsiverelease of TA, which suggests that esterase enzyme can degrade the SMSgels in an on-demand manner to release the encapsulated drugs (FIG. 12).

Example 6

We have investigated the ability of Ascorbic acid palmitate (Asc-Pal,Scheme 6-1) based hydrogels to encapsulate the corticosteroiddexamethasone (Scheme 6-2). Loading efficiency, stability of gels andrelease kinetics in response to enzymes was been investigated.

Encapsulation of Dexamethasone in GRAS-Hydrogels.

Dexamethasone is a hydrophobic drug that exhibits low water solubility.Dexamethasone encapsulated within self-assembled Asc-Pal hydrogels (20%ethanol as co-solvent) demonstrated efficient encapsulation (Table 6-1).Self-assembled fibers were isolated from dexamethasone encapsulatedAsc-Pal hydrogels using multiple cycles of vortex and PBS washes.Isolated fibers were dissolved in DMSO, and the concentration ofdexamethasone was measured using HPLC.

TABLE 6-1 Encapsulation of dexamethasone in Asc-Pal based hydrogels.Amphiphile (Asc-Pal) Encapsulation of concentration, dexamethasone,Solvent % (wt/v) Efficiency (%) 20% Ethanol in water 3 47.67% 20%Ethanol in water 6 62.30%

Self-assembly of the amphiphiles is a delicate process that could beaffected by additional agents. To understand the affect ofdexamethasone, gelation of Asc-Pal has been explored in 20% ethanol inwater by varying concentration of Asc-Pal (1-5% wt/v) while keepingconcentration of dexamethasone (2 mg) constant. Results in Table 6-2reveal that presence of dexamethasone facilitates the self-assembly ofAsc-Pal and strengthen the gelation.

TABLE 6-2 Gelation of Asc-Pal at different concentrations in 20% ofethanol in water without and with (2 mg) of dexamethasone. GelPercentage Without drug With dex (2 mg) 1% Not gel Weak 2% weak Gel 3%Gel Gel 4% Gel Gel 5% Gel Gel

Encapsulation efficiency and loading efficiency were measured from thelyophilized fibers of Asc-Pal gels that were described in Table 6-2.FIGS. 13A-13B reveal that encapsulation and drug loading efficiencieswere increased as the percentage of amphiphile decreased.

Morphology of Dexamethasone Encapsulated Self-Assembled Hydrogels.

Scanning electron microscope images revealed that dexamethasoneencapsulated Asc-Pal hydrogels consist of fibrous morphology (up tomicron width and several microns length) that is similar to Asc-Pal gelwithout dexamethasone.

On-Demand Delivery of Dexamethasone from Gras-Based Asc-PalSelf-Assembled Hydrogels.

Different amounts of dexamethasone (2 and 4 mg) in 5% (wt/v) of Asc-Palself-assembled gels (ethanol:water, 1:3) were subjected to controlledrelease experiments. In the absence of the esterase enzyme,dexamethasone has not been released, a moderate burst release (<10%) hasbeen observed. On contrary, presence of lipase enzyme triggered therelease of encapsulated dexamethasone. Interestingly, different startingamounts of dexamethasone gels have shown similar release profiles. FIGS.14A-14B show the controlled release of dexamethasone from self-assembledhydrogels (ethanol:water, 1:3) of Asc-Pal (5%, wt/v) in the absence andpresence of esterase enzyme (10,000 units) at 37° C. Controlled releasewas examined for FIG. 14A: 2 mg and FIG. 14B: 4 mg of dexamethasoneencapsulated within Asc-Pal hydrogels. Similar release profile has beenobserved from both gels.

The preformed Asc-Pal hydrogel was degraded completely by the lipase (anesterase) while releasing the encapsulated dexamethasone. As enzymeresponsive release of dexamethasone occurred, it is important tounderstand the degradation products from Asc-Pal hydrogels. Asc-Palamphiphiles encompass an ester bond that connects ascorbic acid andpalmitic acid (FIGS. 15A-15B). Thus it is anticipated that lipasedegrades the hydrogel by hydrolyzing ester bonds of Asc-Pal amphiphile.The formation of byproduct ascorbic acid was followed by HPLC. To followthe formation of ascorbic acid two-sets of Asc-Pal hydrogels (3 and 6%wt/v) were made with 0.42 (FIG. 15A) and 1 (FIG. 15B) mg ofdexamethasone loading. These hydrogels were subjected to lipase enzyme,enzyme responsive formation of ascorbic acid (FIGS. 15A-15B) reveal thatAsc-Pal hydrogels degrade in response to the enzyme that cleavage esterbonds in the amphiphilic gelators by the hydrolase enzyme.

Addition of excipients to self-assembled hydrogels can influence thegelation ability. Strength of the hydrogels can be altered throughaddition of an appropriate excipient during the self-assembly process.Addition of an excipient could be used to alter the release of theencapsulated agents in addition to altering gelation strength. Tomodulate the gelation strength of Asc-Pal hydrogels (3 and 6%)glycocholic acid (1 mg) was added during self-assembly process, andthese gels were subjected to lipase mediated degradation. Interestingly,data in FIG. 16 reveal that addition of glycocholic acid reversed theenzyme responsiveness. Presence of lipase reduced the release ofdexamethasone compared to the control (PBS, FIG. 16). The rigidsteroidal backbone of glycocholic acid may induce destabilization of theself-assembled lamellar structures of Asc-Pal amphiphile. Thus doping ofglycocholic acid may perturb optimal encapsulation of dexamethasone thatresult in a non-enzymatic responsive release of dexamethasone.

Effect of Dexamethasone Structure on its Release from Asc-Pal Hydrogels.

Hydrophobic analogue of dexamethaonse, i.e., palmitated dexamethasone(Dex-Pal) was used to incorporate Dex-Pal within self-assembled fibersof Asc-Pal. Rationale for using dexamethasone palmitate is that thepresence of a hydrophobic chain facilitates efficient incorporation ofhydrophobic drugs within self-assembled lamellar structures of Asc-Pal.To elucidate release kinetics, 1.6 mg of Dex-Pal has been encapsulatedin 16 mg of Asc-Pal in 200 μl of 20% DMSO/water. In the absence of theesterase enzyme, upon incubation in PBS at 37° C.,dexamethasone-palmitate encapsulated Asc-Pal hydrogels did not releasedexamethasone (FIG. 17). Even after 11 days, there was no significantamount of dexamethasone released from these gels. However, at day 11,lipase enzyme promoted degradation of the gels to release dexamethasonein an on-demand manner (FIG. 17).

Example 7

We have investigated the ability of Ascorbic acid palmitate (Asc-Pal),gels to encapsulate the anti-inflammatory agent, Indomethacin (Scheme7-1). Loading efficiency, stability of gels and release kinetics inresponse to the enzymes has been investigated. Detailed results havebeen summarized in following sections.

Scheme 7-1.

Chemical structure of non-steroidal anti-inflammatory drug (NSAID),indomethacin.

Controlled Release of Indomethacin.

Non-steroidal anti-inflammatory drug, indomethacin has been encapsulatedin Asc-Pal gels with high loading efficiency (˜83%). Release kineticexperiments reveal that indomethacin encapsulated Asc-Pal gel does notexhibit burst release (FIG. 18). In the absence of enzymes, these gelswere stable and showed only moderate release of indomethacin (˜20%) andreached a plateau within ˜24 hr. On the contrary, in the presence of anesterase enzyme (lipase, 10,000 units), the gels exhibitenzyme-responsive release of TA. This demonstrates that enzymes candegrade the SMS gels in an on-demand manner to release the encapsulateddrugs (FIG. 18).

Example 8

Self-assembled nanofibrous hydrogels of SMS amphiphiles have been usedfor encapsulating small interference RNA (siRNA). In this instance, GL3siRNA (sense: 5′-CUU ACG CUG AGU ACU UCG AdTdT-3′ (SEQ ID NO:1) andantisense: 5′-UCG AAG UAC UCA GCG UAA GdTdT-3′ (SEQ ID NO:2)) is knownto silence firefly luciferase in cells. GL3 encapsulated hydrogel hasshown fibrous-like morphology. To quantify the encapsulation efficiency,fluorescent dye (Cy-3) labeled DNA with same sequence as GL3 was chosento mimic similar charge that can influence encapsulation. Afterencapsulating the siRNA into SMS hydrogels, inherent gelation propertiessuch as minimum gelation concentration and gel stability have not beenperturbed by presence of siRNA. Quantification of encapsulated Cy3-DNArevealed that SMS hydrogels entrapped DNA with 15% encapsulationefficiency. However, further optimization of protocols couldsignificantly enhance the encapsulation efficiency. DNA encapsulatedself-assembled fibers were washed with phosphate buffered saline toremove non-encapsulated DNA. Subsequently, DNA encapsulated fibers weredissolved in DMSO and presence of DNA has been quantified usingabsorbance spectra (FIG. 19). Referring to FIG. 19, the presence of anabsorption peak at 555 nm for DNA-encapsulated SMS hydrogel compared toonly SMS hydrogel clearly suggests that DNA has been encapsulated withinthe gel.

Example 9

We have investigated the ability of GRAS-amphiphiles, Ascorbic acidpalmitate (Asc-Pal, Sorbitan monostearate (SMS) and Triglycerolmonostearate (TG-18, Scheme 9-1) based hydrogels to encapsulate insulin.Loading efficiency, stability of gels and morphology of the gels wasbeen investigated.

Encapsulation of Insulin in GRAS-Hydrogels.

Insulin was encapsulated within self-assembled Asc-Pal or SMS hydrogelsdemonstrated efficient encapsulation between 58-80%. Self-assembledparticles were isolated from insulin encapsulated SMS hydrogels usingmultiple cycles of vortex and PBS washes. Isolated particles weredissolved, and the concentration of insulin was measured using Bradfordassay. Loading efficiency of insulin in GRAS-based hydrogels was 15-35%(wt/wt).

Morphology of Insulin Encapsulated SMS Hydrogels.

Insulin encapsulated SMS hydrogels were characterized under scanningelectron microscope (SEM). Hydrogels were tested as native gels,lyophilized xerogels and xerogels in the presence of stabilizers such astrehalose and Tween-20. These hydrogels showed particle-like morphologyunder scanning electron microcopy.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A self-assembled gel composition controllablyreleasing an agent comprising a gel composition prepared byself-assembly of a homogeneous solution of an enzyme cleavableamphiphilic gelator having a molecular weight of 2500 or less in aconcentration of greater than 3 wt/vol % in one or more aqueous solventscontaining an organic solvent in a concentration of between 0.5 and 75vol/vol % of the total solvent volume, wherein the gelator meets therequirements of the FDA for Generally Recognized As Safe compounds; andone or more agents for delivery from the gel composition; wherein theself-assembled gel composition comprises nanostructures comprisinglamellar structures, fibers, sheet-like structures, tape-likestructures, nanoparticles, or combinations thereof, each comprising theenzyme cleavable amphiphilic gelator; wherein the one or more agents areat least partially encapsulated or entrapped by the nano structures, andwherein the agent is released from the gel composition in the presenceof enzyme, and wherein the self-assembled gel is a viscous homogenousgel stable to inversion, optionally wherein solvent is removed from thegel composition by centrifugation, washing, lyophilization, drying, orcombinations thereof.
 2. The self-assembled gel composition of claim 1wherein the enzyme cleavable amphiphilic gelator is an ascorbylalkanoate selected from the group consisting of ascorbyl palmitate,ascorbyl decanoate ascorbyl laurate, ascorbyl caprylate, ascorbylmyristate, ascorbyl oleate, and combinations thereof.
 3. Theself-assembled gel composition of claim 2, wherein the ascorbylalkanoate is ascorbyl palmitate.
 4. The self-assembled gel compositionof claim 1, wherein the enzyme cleavable amphiphilic gelator is asorbitan alkanoate selected from the group consisting of sorbitanmonostearate, sorbitan decanoate, sorbitan laurate, sorbitan caprylate,sorbitan myristate, sorbitan oleate, and combinations thereof.
 5. Theself-assembled gel composition of claim 4, wherein the sorbitanalkanoate is sorbitan monostearate.
 6. The self-assembled gelcomposition of claim 1, wherein the enzyme cleavable amphiphilic gelatoris a triglycerol monoalkanoate selected from the group consisting oftriglycerol monopalmitate, triglycerol monodecanoate, triglycerolmonolaurate, triglycerol monocaprylate, triglycerol monomyristate,triglycerol monostearate, triglycerol monooleate, and combinationsthereof.
 7. The self-assembled gel composition of claim 6, wherein thetriglycerol monoalkanoate is triglycerol monopalmitate.
 8. Theself-assembled gel composition of claim 1, wherein the enzyme cleavableamphiphilic gelator is a sucrose alkanoate selected from the groupconsisting of sucrose palmitate, sucrose decanoate, sucrose laurate,sucrose caprylate, sucrose myristate, sucrose oleate, and combinationsthereof.
 9. The self-assembled gel composition of claim 8, wherein thesucrose alkanoate is sucrose palmitate.
 10. The self-assembled gelcomposition of claim 1, wherein the enzyme cleavable amphiphilic gelatoris glycocholic acid.
 11. A self-assembled gel composition controllablyreleasing an agent comprising a gel composition prepared byself-assembly of a homogeneous solution containing at least 3 wt/vol %of an enzyme cleavable amphiphilic gelator having a molecular weight of2500 or less, a non-independent gelator, in one or more aqueous solventscontaining an organic solvent in a concentration of between 0.5 and 75vol/vol % of the total solvent volume, wherein the gelator meets therequirements of the FDA for Generally Recognized As Safe compounds; andone or more agents for delivery from the gel composition after exposureto enzyme under physiological conditions; wherein the enzyme cleavableamphiphilic gelator and the non-independent gelator in one or moreaqueous solvents containing an organic solvent in a concentration ofbetween 0.5 and 75 vol/vol % of the total solvent volumeco-self-assemble into a viscous homogeneous gel, wherein the gelcomposition is stable to inversion and comprises nanostructurescomprising lamellar structures, fibers, sheet-like structures, tape-likestructures, nanoparticles, or combinations thereof, each comprising theenzyme cleavable amphiphilic gelator and the non-independent gelator;wherein the one or more agents are at least partially encapsulated orentrapped by the nano structures; and wherein solvent is removed fromthe gel composition by centrifugation, washing, lyophilization, drying,or combinations thereof.
 12. The self-assembled gel composition of claim1, wherein the solvent is removed completely and the self-assembled gelcomposition is in the form of a powder or powder formulation.
 13. Theself-assembled gel composition of claim 1, from which all of the solventhas been removed.
 14. The self-assembled gel composition of claim 1,wherein the organic solvent is selected from the group consisting ofbenzene, toluene, carbon tetrachloride, acetonitrile, glycerol,1,4-dioxane, dimethyl sulfoxide, ethylene glycol, methanol, chloroform,hexane, acetone, N, N′-dimethyl formamide, ethanol, and combinationsthereof.
 15. The self-assembled gel composition of claim 1, wherein theone or more agents are selected from the group consisting of peptides,proteins, nucleic acids, polynucleotides, small molecule agents, andcombinations thereof.
 16. The self-assembled gel composition of claim 1,wherein the one or more agents are selected from the group consisting ofanti-inflammatory agents, chemotherapeutics, PARP-inhibitors, steroids,vitamins, anti-pain agents, anti-pyretic agents, anti-depression agents,vasodilators, vasoconstrictors, immune-suppressants, tissue regenerationpromoters, combinations thereof.
 17. The self-assembled gel compositionof claim 1, wherein the one or more agents are selected from the groupconsisting of dexamethasone, temozolomide, triamcinolone acetonide,camptothecin, iodomethacin, paclitaxel, carmustine, curcumin, cisplatin,BMS-536924, ethambutol, insulin,1,10-Dioctadecyl-3,3,30,30-tetramethylindodicarbocyanine4-chlorobenzenesulfonate salt (DiD) dye, Cy3 dye, and small interferingRNA (siRNA).
 18. The self-assembled gel composition of claim 16, whereinthe PARP-inhibitors are selected from the group consisting of NU1025,BSI-201, AZD-2281, ABT-888, AGO-14699, 4-hydroxyquinazoline,3-aminobenzamide, 1,5-isoquinolinediol, 4-amino-1,8-naphthalimide, andO⁶-benzylguanine.
 19. The self-assembled gel composition of claim 1,wherein the agent is a chemotherapeutic agent.
 20. The self-assembledgel composition of claim 19, wherein the chemotherapeutic agent istemozolomide, carmustine, camptothecin, or paclitaxel.
 21. Theself-assembled gel composition of claim 1, wherein the enzyme cleavableamphiphilic gelator having a molecular weight of 2500 or less isselected from the group consisting of ascorbyl alkanoate, sorbitanalkanoate, triglycerol monoalkanoate, sucrose alkanoate, glycocholicacid, and combinations thereof.
 22. The self-assembled gel compositionof claim 1, further comprising a non-independent gelator.
 23. Theself-assembled gel composition of claim 1, wherein the nanostructuresare lamellar structures.
 24. The self-assembled gel composition of claim1 wherein the enzyme cleavable amphiphilic gelator and the one or moreaqueous solvents are mixed with heating and the viscous homogeneous gelforms within 15 to 45 minutes of cooling.
 25. The self-assembled gelcomposition of claim 1 comprising 4 wt/vol % or more of the enzymecleavable amphiphilic gelator.
 26. The self-assembled gel composition ofclaim 1 wherein in the absence of enzyme, the agent is released with aminimal burst of less than 20% of agent in the self-assembled gelcomposition measured at 37° C. in phosphate buffered saline.
 27. Theself-assembled gel composition of claim 1 wherein the enzyme cleavableamphiphilic gelator is dissolved in a mixture of water and the organicsolvent to form a stable gel in the absence of buffer.
 28. Theself-assembled gel composition of claim 1 in the form of an inhalable orintranasal dried powder.
 29. The self-assembled gel composition of claim1 in the form of dry powder incorporated into a lozenge or chewing gum.30. The self-assembled gel composition of claim 11, wherein thenon-independent gelator is selected from the group consisting of alphatocopherol acetate, retinyl acetate, and retinyl palmitate.
 31. Theself-assembled gel composition of claim 11 wherein, in the absence ofenzyme, the agent is released with a minimal burst of less than 20% ofthe agent in the self-assembled gel composition measured at 37° C. inphosphate buffered saline.
 32. The self-assembled gel composition ofclaim 1, wherein the organic solvent is in a concentration of between0.5 and 30 vol/vol % of the solvent volume in the homogeneous solution.33. The self-assembled gel composition of claim 11, wherein the organicsolvent is in a concentration of between 0.5 and 30 vol/vol % of thesolvent volume in the homogeneous solution.